Device and methods for epigenetic analysis

ABSTRACT

Provided herein are methods and devices for single object detection. The methods and devices can be used to identify a plurality epigenetic markers on a genetic material, or a chromatin, encompassing fragments thereof. The invention provides for the characterization of the genetic material flowing through a channel in a continuous body of fluid based on detection of one or more properties of the genetic material. The methods and systems provided herein allow genome-wide, high-throughput epigenetic analysis and overcome a variety of limitations common to bulk analysis techniques.

CROSS-REFERENCE

This application is a continuation-in-part of PCT Patent Application No.PCT/US2010/044806, filed Aug. 6, 2010, which claims benefit of priorityto U.S. Provisional Application No. 61/231,979, filed Aug. 6, 2009, U.S.Provisional Application No. 61/307,827, filed Feb. 24, 2010, U.S.Provisional Application No. 61/231,963, filed Aug. 6, 2009, and U.S.Provisional Application No. 61/359,266, filed Jun. 28, 2010; thisapplication is also a continuation-in-part of PCT Patent Application No.PCT/US2010/044810, filed Aug. 6, 2010, which claims benefit of priorityto U.S. Provisional Application No. 61/231,979, filed Aug. 6, 2009, U.S.Provisional Application No. 61/307,827, filed Feb. 24, 2010, U.S.Provisional Application No. 61/231,963, filed Aug. 6, 2009, and U.S.Provisional Application No. 61/359,266, filed Jun. 28, 2010, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Contract number R01DA025722 by the National Institutes of Health,Contract number ECS-9876771 by the National Science Foundation (NBTC),and Contract number 199-3166 by the Center for Vertebrate Genomics(Cornell University).

BACKGROUND OF THE INVENTION

Chromatin, located in the nucleus of eukaryotic cells, is the complex ofDNA with histone proteins, including, H1, H2A, H2B, H3, and H4. Thehistone proteins are assembled onto DNA in the form of a nucleosome(Luger et al. (1997) Nature, 389, 251-260). The DNA sequence carries thegenetic code and controls inheritance of traits. However, reversiblecovalent modifications to specific DNA sequences and their associatedhistones can influence how the underlying DNA is utilized and cantherefore also control traits (Jenuwein and Allis (2001) Science, 293,1074-1080; Klose and Bird (2006) Trends In Biochemical Sciences, 31,89-97). These have been referred to as epigenetic modifications. Themost common epigenetic modifications to DNA in mammals are methylationand hydroxymethylation of DNA, either of which may be placed on thefifth carbon of the cytosine pyrimidine ring. A host of modificationsincluding methylation, acetylation, ribosylation, phosphorylation,sumoylation (related to small ubiquitin-like modifiers), ubiquitylationand citrullination can occur at more than 30 amino acid residues of thefour core histones within the nucleosome. Epigenetic modifications tothe mammalian genome include methylation of cytosines in the DNA;methylation, acetylation, ribosylation, phosphorylation, sumoylation andubiquitylation of the histones bound to the DNA; and the precisepositioning of histone containing nucleosomes over the DNA.

Epigenetic alterations to the genome can influence development andhealth as profoundly as mutagenesis of the genome. One of the mostdramatic examples is the methylation of DNA at the promoter of the p16tumor suppressor. Such methylation silences the gene, and so domutations to the gene sequence itself, and both events contribute to thedevelopment and progression of colorectal cancer. However, unlikemutations, epigenetic silencing of p16 can be reversedpharmacologically, and hence providing the possibility of medicalintervention.

Specific changes in epigenetic state that occur genome wide appear toregulate cellular differentiation during development (Mikkelsen et al.(2007) Nature, 448, 553-U552). Perturbations of normal epigenetic statein mature tissues contribute to initiation and progression of cancer andother diseases (Feinberg, A. P. (2007) Nature, 447, 433-440).Additionally, studies have shown that epigenetic states are influencedby environmental variables including diet (Waterland and Jirtle (2003)Molecular And Cellular Biology, 23, 5293-5300), environmental toxins(Anway et al. (2005) Science, 308, 1466-1469) and maternal behaviors(Weaver et al. (2004) Nature Neuroscience, 7, 847-854). Given thefundamental role that epigenetic mechanisms play in normal development,environmental responses and how their perturbation affects diseasestate, there is increasing effort devoted to characterizing the humanepigenome (Bernstein et al. (2007) Cell, 128, 669-681).

These epigenetic modifications do not alter the primary DNA sequence,but they have a potent influence on how those underlying DNA sequencesare expressed. As a result, changes in epigenetic state can alterphenotypes as powerfully as alterations in DNA sequence. Also like DNAsequence states, epigenetic states can be passed from mother to daughtercells during mitosis, and can even persist through meiosis to betransmitted from one generation to the next. Although epigenetic markscan change and revert to their original state far more readily thanchanges in DNA sequence, they are as fundamental to development anddisease as the nature of the DNA sequences on which they reside.

Abundant evidence has demonstrated the importance of epigeneticregulation to human disease—notably cancer. Early observations linkedperturbations in DNA methylation to the development of human colorectalcancer and subsequent studies showed that experimental manipulation ofDNA methylation state, pharmacologically or genetically, altered tumordevelopment. These have motivated studies that correlate epigenomicprofiling of tumor specimens with disease state and clinical outcomes ofthe individuals providing the specimens. Importantly, ongoing clinicaltrials using drugs that modify epigenetic states have shown therapeuticpromise and the ability to attenuate epigenetic biomarkers that indicatepoor prognoses. Although changes in DNA methylation during cancerdevelopment and progression have attracted considerable attention, DNAmethylation states are also influenced by alterations in histonemodification state and nucleosome positioning. The reciprocalinteractions among all these epigenetic marks are all likely to be ofimportance to cancer development and progression.

In addition to controlling processes fundamental to cancer, epigeneticstates influence responses of mammals to changes in their environment.Maternal behavior during nursing, exposure to endocrine disruptors andthe nutrient composition of diets each have been shown to elicitspecific phenotypes that correlate with specific changes in epigeneticstates. Most importantly, these phenotypes, and the accompanyingepigenetic alterations, can be transmitted from parent to offspring,even if only the parents and not the offspring experienced theenvironmental insult. This raises the possibility that some complextraits that run in families, like obesity, cancer or behavioralpatterns, are transmitted by epigenetic means and result fromenvironmental exposures experienced during prior generations.

A common theme in biology is that mechanisms influencing disease stateslike cancer, or homeostatic responses to environment are alsofundamental to development. Epigenetic mechanisms have been shown to becritical to Drosophila development and mammalian development.

Existing approaches for analyzing epigenetic modifications of chromatin,such as chromatin immunoprecipitation (ChIP), can evaluate only oneepigenetic mark at a time and are labor-intensive, serial processes thatimpose significant limitations on analysis throughput and samplequantity. The ChIP technique involves immunoprecipitation using anantibody specific to one epigenetic modification of interest to isolatemodified chromatin, which is subsequently analyzed using massivelyparallel DNA sequencing, microarray hybridization or gene-specific PCR.This method can be used to characterize the genome placement of achromatin associated protein and is the predominant analytical toolcurrently practiced in epigenomic and chromatin research. However, itsuffers from two major limitations. First, the analysis requires 10⁴ to10⁷ cells and is incapable of assessing epigenetic changes in vanishingquantities, making studies of developing embryos, sorted cells ormicrodissected cells impossible. Second, only one epigenetic mark can beisolated at a time, making detection of co-existent marks verydifficult. Measurements provide only information about the ‘average’chromatin state in a cell population and nothing about the individualDNA strands. In one published example involving the characterization ofbivalent states in ES cells, these limitations introduced ambiguity asto if tri-methylated histone H3 lysine 27 (H3K27me3) and tri-methylatedhistone H3 lysine 4 (H3K4me3) marks were present simultaneously on agiven gene or if two populations existed with the ES culture, each witha mutually exclusive mark. Sequential ChIPs against two differentantibodies may circumvent this limitation, however this solution isimpractical for whole genome analysis.

As such, current methods for epigenomic testing involve bulk moleculeanalysis. The results are representative of a composite signal frommultiple copies of a genetic material, which is distinct from singlemolecule analysis. Histone modifications are most commonly detectedusing chromatin immunoprecipitation (ChIP), in whichmodification-specific antibodies are used to immunoprecipitate theassociated DNA, which is then detected by hybridization to microarray(Ren et al. (2000) Science, 290, 2306-2309) (ChIP-chip) or deepsequencing (Barski et al. (2007) Cell, 129, 823-837) (ChIP-seq). DNAmethylation can also be detected by immunoprecipitation using amethylcytosine antibody (Weber et al. (2005) Nature Genetics, 37,853-862), or with bisulfite sequencing, which offers more comprehensiveanalysis of DNA methylation states (Zhang et al. (2006) Cell, 126,1189-1201). Genome wide epigenomic analyses using antibodies often useon the order of 10⁶ to 10⁷ cells. ChIP has been used with as few as 100cells, however, with this few cells the analysis was locus specific andnot genome wide (O'Neill et al. (2006) Nature Genetics, 38, 835-841). Afar more significant limitation is encountered when studies seek todetermine whether or not two or more epigenetic marks are coincidentwithin the genome or are present on a single piece of genetic material,e.g., a single chromatin. Analysis of each epigenetic mark requires anindependent immunoprecipitation. When precipitating chromatin from anensemble of cells with different antibodies, it is difficult todistinguish true coincidence of the detected marks from the existence ofmultiple populations within the ensemble, each with a differentepigenomic profile. This can be somewhat overcome with sequential ChIP,where the material precipitated by one antibody is re-ChIPed with asecond antibody (Bernstein et al. (2006) Cell, 125, 315-326). However,these techniques are not amenable to genome wide analysis or for studiesin which more than two epigenetic marks are investigated. Furthermore,bulk analysis techniques report an average of the population and do notconsider variations at the single molecule level. Thus, there remains aconsiderable need for alternative methods and compositions that providefor a more robust genome-wide epigenetic analysis. The present inventionsatisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides for assessingepigenetic variations at a single chromatin level. Single-moleculeanalysis provides several advantages over conventional approaches.First, the analysis provides information on individual molecules whoseproperties are hidden in the statistically averaged information that isrecorded by ordinary ensemble measurement techniques. In addition,because the analysis can be multiplexed, it is conducive tohigh-throughput implementation, requires smaller amounts of reagent(s),and takes advantage of the high bandwidth of detection systems such asmodern avalanche photodiodes for extremely rapid data collection.Moreover, because single-molecule counting automatically generates adegree of immunity to illumination and light collection fluctuations,single-molecule analysis can provide greater accuracy in measuringquantities of material than bulk techniques. As such, single-moleculeanalysis greatly improves the efficiency and accuracy in epigeneticanalysis, genotyping, gene expression profiling, DNA sequencing,nucleotide polymorphism detection, pathogen detection, proteinexpression profiling, and drug screening. Therefore, there is aconsiderable need to overcome these disadvantages of bulk analysistechniques. Accordingly, the present invention provides devices andmethods that should overcome the limitations of the prior art andtransform how epigenetic and chromatin research is performed.

The present invention provides for methods for performing epigeneticanalysis of a genetic material in a channel, comprising: (a) flowing thegenetic material through said channel, wherein said genetic material islabeled with a plurality of labels, at least one of which isspecifically complexed with an epigenetic marker on said geneticmaterial, and at least one other label is complexed with a proteinand/or nucleotide of said genetic material; (b) illuminating the channelto create a plurality of interrogation volumes, each of which isconfined by walls of said channel and a beam of light; and (c) detectingthe at least one label and the one other label from the same or distinctinterrogation volumes of said plurality to generate time-correlatedresolution of said at least one and said at least one other label,thereby performing said epigenetic analysis.

Another aspect of the invention provides for methods for performingepigenetic analysis of a genetic material in a channel, comprising: (a)flowing the genetic material through an illuminated interrogationvolume, said interrogation volume being confined by walls of saidchannel and a beam of light, wherein said genetic material is labeledwith a plurality of labels, at least one of which is specificallycomplexed with an epigenetic marker on said genetic material, and atleast one other label is complexed with a protein and/or nucleotide ofsaid genetic material; and (b) simultaneously detecting the at least onelabel and the at least one other label within the interrogation volume,thereby performing said epigenetic analysis.

A further aspect of the invention provides for methods for obtainingreal-time analysis data regarding a material in a channel, comprising:(a) flowing the material through an illuminated interrogation volume,said interrogation volume being confined by walls of said channel and abeam of light, wherein said material is labeled with a plurality oflabels, at least one of which is specifically complexed with a marker onsaid material, and at least one other label is complexed with saidmaterial; (b) detecting the at least one label and the at least oneother label within the interrogation volume in real-time, therebyproducing said real-time analysis data; and (c) displaying saidreal-time analysis data on a display device, wherein said real-timeanalysis data reflects information on said at least one label and saidat least one other label.

One aspect of the invention provides for a system for characterizing asingle nucleic acid molecule, comprising: (a) a channel configured tohold said molecule; (b) one or more light sources configured toilluminate the channel to create one or more interrogation volumes; (c)a detection system configured to detect at least two types of signalsindicative of two distinct properties of said nucleic acid molecule fromthe one or more interrogation volumes; and (d) a processor programmed toprovide real-time time-correlated resolution of the at least two typesof signals from said one or more interrogation volumes, therebycharacterizing said single nucleic acid molecule.

In a further aspect of any of the foregoing systems and methods, the twotypes of signals are detected simultaneously or the detection systemdetects four types of signals. In a further aspect of any of theforegoing systems and methods, the processor is characterized in that:(i) it is programmed to provide time-correlating resolution of the atleast two types of signals from more than one interrogation volume; or(ii) comprises a field-programmable gate array programmed to providereal-time time-correlated resolution of the at least two types ofsignals.

Another aspect of the invention provides for a system for characterizingan object, comprising: (a) a channel configured to hold said object; (b)one or more light sources configured to illuminate the channel to createone or more interrogation volumes; (c) a detection system configured todetect at least two types of signals indicative of two distinctproperties of said object from the one or more interrogation volumes;(d) a processor programmed to provide real-time time-correlatedresolution of the at least two types of signals from said one or moreinterrogation volumes, thereby characterizing said object; and (e) auser interface configured to receive data from the processor and displaythe data, wherein the data comprises information on the at least twotypes of signals from said one or more interrogation volumes.

The present invention provides for methods comprising the steps of a)providing a sample comprising DNA fragments or chromatin, wherein thechromatin comprises fragments of DNA; b) labeling said DNA fragments orchromatin with one or more labels specific for DNA; c) labeling said DNAfragments or chromatin with one or more labels specific for one or moreepigenetic markers; d) contacting the labeled sample with a microfluidicdevice comprising a submicrometer channel; e) applying a voltagepotential or pressure differential across the submicrometer channel,wherein the voltage potential or pressure differential is sufficient toflow said DNA fragments or chromatin through the submicrometer channel;f) detecting DNA by detecting the one or more labels specific for DNA;and g) detecting epigenetic markers by detecting the one or more labelsspecific for one or more epigenetic markers; wherein the detection isperformed with single molecule resolution.

The present invention provides methods comprising contacting a devicecomprising a submicrometer channel comprising a detection volume and afirst end and at least a second end with a reaction mixture comprising(a) a plurality of chromatin fragments comprising DNA and one or moreepigenetic modifications; (b) a label specific for DNA; and (c) a labelspecific for the one or more epigenetic modifications; wherein thesubmicrometer channel is of a size that essentially one chromatinfragment is disposed within the detection volume at a time.

In one embodiment, the invention provides for methods of flowing a DNAsample in a solution confined within a nanofluidic channel, illuminatingthe sample with a Gaussian shaped laser profile, and detectingfluorescence events indicative of histone or DNA components. The samplemay be illuminated with two Gaussian shaped laser profiles. In someembodiments, the light source for illuminating the sample is an LED or aVCSEL.

A further aspect of the invention provides for time coincident detectionof (a) one or more labels specific for DNA; and (b) one or more labelsspecific for one or more epigenetic modifications.

In any of the foregoing aspects and embodiments, the genetic material isa chromatin, a nucleic acid molecule, or a single nucleic acid molecule.The material to be analyzed and/or sorted can be a chromatin, a nucleicacid molecule, or a single nucleic acid molecule.

In any of the foregoing aspects and embodiments, the material can beanalyzed with single molecule resolution. In some embodiments, thedetecting step provides a time-dependent resolution of better than about10 microseconds. The subject methods can further comprise detecting timecoincident detection of one or more labels specific for DNA and one ormore labels specific for one or more epigenetic markers. The one or moreepigenetic markers can be selected from one or more methylated DNAnucleotides, one or more hydroxymethylated DNA nucleotides, one or moreacetylated histones, one or more methylated histones, one or moreubiquinated histones, one or more sumoylated histones, one or morephosphorylated histones, methyl DNA binding protein (MDB1), RNApolymerase II, and SWI/SNF.

In any of the foregoing aspects and embodiments, the one or moremethylated DNA nucleotides can comprise 5-methylcytosine. The one ormore acetylated histones can comprise acetylated histone H3 K4,acetylated histone H3 K9, acetylated histone H3 K14, acetylated histoneH3 K18, or acetylated histone H3 K23. The one or more acetylatedhistones can comprise acetylated histone H4 K5, acetylated histone H4K8, acetylated histone H4 K12, or acetylated histone H4 K16. The one ormore methylated histones may comprise mono, di, or tri methylatedhistone H3 K4; mono, di, or tri methylated histone H3 K9; mono, di, ortri methylated histone H3 K27; or mono, di, or tri methylated histone H3K36. The one or more methylated histones may comprise mono, di, or trimethylated histone H4 K20. The one or more methylated histones maycomprise mono or di methylated histone H3 R2; mono or di methylatedhistone H3 R17; mono or di methylated histone H3 R26; mono or dimethylated histone H3 R128; mono or di methylated histone H3 R129; monoor di methylated histone H3 R131; or mono or di methylated histone H3R134. The one or more methylated histones may comprise mono or dimethylated histone H4 R3.

In some cases the epigenetic modifications are selected from the groupconsisting of methylated DNA nucleotides (e.g. 5-methylcytosine),hydroxymethylated DNA nucleotides, acetylated histones (e.g. H3 K4, H3K9, H3 K14, H4 K12, H3 K18, H3 K23, H4 K5, H4 K8, H4 K12, H4 K16),methylated histones (e.g. mono di or tri methylated H3 K4, H3 K9, H3K27, H3 K36, H4 K20, H3 R2, H3 R17, H3 R26, H3 R128, H3 R129, H3 R131,H3 R134, H4 R3), ubiquinated histones, sumoylated histones,phosphorylated histones, the presence of methyl DNA binding protein, thepresence of SWI/SNF, and the presence of RNA polymerase II.

In a further aspect of any one of the foregoing embodiments, the presentinvention provides identifying the time coincident detection of (a) oneor more labels specific for DNA; and (b) one or more labels specific forone or more epigenetic modifications as indicating epigeneticallymodified DNA or chromatin.

In a further aspect of any one of the foregoing aspects and embodiments,the methods may further comprise determining whether a time coincidentor time-correlated detection of the label specific for DNA and the labelspecific for the one or more epigenetic modifications has occurred. Insome embodiments, the methods provided herein may further compriseidentifying the time coincident detection of the one or more labelsspecific for DNA and the one or more labels specific for one or moreepigenetic markers as indicating epigenetically modified DNA. Themethods may further comprise identifying the time coincident detectionof the label specific for DNA and the label specific for the one or moreepigenetic modifications as the detection of an epigenetically modifiedchromatin fragment. The methods may further comprise counting the numberof epigenetically modified chromatin fragments detected.

In a further aspect of any of the foregoing aspects and embodiments, themethods may comprise detecting at least three or four labels.

In a further aspect of any of the foregoing aspects and embodiments, themethods can comprise detecting one label that is specifically complexedwith an epigenetic marker and at least one other label. The at least oneother label can be complexed with a histone or is a nucleic acid bindingagent selected from the group consisting of sequence specific probe,intercalating dye, minor groove binder, and DNA binding proteins. The atleast one other label is complexed with a binding agent that complexeswith a target selected from the group consisting of a non-histoneprotein, a transcription factor, MBD1, RNA Pol II, and RNA.

In any of the foregoing aspects and embodiments, the interrogationvolume(s) is/are less than about 10, 1, 0.5, 0.2, 0.1, or 0.05femtoliters. The genetic material can be characterized in less thanabout 1, 0.5, 0.1, 0.01, 0.001, or 0.0001 seconds. The illuminatedinterrogation volume(s) can contain a single chromatin. The one or moreinterrogation volumes can be one or more of (a) less than about 10, 1,0.5, 0.2, 0.1, or 0.05 femtoliters, (b) no greater than about 10,000times of the size of the object, provide single molecule resolution, (c)dimensioned to hold a single nucleic acid molecule, and (d) confined bythe walls of the channel and a beam of light from said light sources,said beam of light having a diameter no greater than X microns.

In any of the foregoing embodiments, the detection system can measure asignal to noise ratio of greater than about 2. The channel comprises anoptically transparent wall. The light sources provide a plurality oflight beams of a varying wavelength.

In a further aspect of any one of the foregoing embodiments, the presentinvention provides for excitation of the label specific for the one ormore epigenetic modifications and excitation of the label specific forDNA with an illumination source such as an incandescent source, a lightemitting diode, a flash lamp, or a laser. The illumination source may beone or more lasers. In a further aspect of any one of the foregoingembodiments, the present invention provides for detecting emittedphotons with a photodetector such as a photomultiplier tube, anavalanche photodiode, a CCD, or CMOS array.

The emission may comprise detecting emitted photons with an avalanchephotodiode or a photomultiplier tube.

The methods may further comprise counting the amount of epigeneticallymodified DNA. The methods may further comprise storing the results ofsaid counting the amount of epigenetically modified DNA on a computerreadable medium.

The methods may further comprise storing the number of epigeneticallymodified chromatin fragments detected on a computer readable medium.

The methods may further comprise varying the amount of the appliedvoltage across the submicrometer channel to sort epigenetically modifiedDNA from DNA that does not provide time coincident detection of one ormore labels specific for DNA and one or more labels specific for one ormore epigenetic markers. The methods may further comprise varying theamount of the applied pressure across the submicrometer channel to sortepigenetically modified DNA from DNA that does not provide timecoincident detection of one or more labels specific for DNA and one ormore labels specific for one or more epigenetic markers. The methods mayfurther comprise applying a sorting laser to sort epigeneticallymodified DNA from DNA that does not provide time coincident detection ofone or more labels specific for DNA and one or more labels specific forone or more epigenetic markers. The subject methods may further comprisevarying a magnetic moment across the submicrometer channel to sortepigenetically modified DNA from DNA that does not provide timecoincident detection of one or more labels specific for DNA and one ormore labels specific for one or more epigentic markers.

In any of the foregoing embodiments, the sorted epigenetically modifiedDNA can be further analyzed by PCR, nucleic acid hybridization analysis,microarray analysis, ChIP, or DNA sequencing. In any of the foregoingembodiments, the methods may further comprise calculating the size ofthe detected DNA from a signal magnitude provided by the detecting ofthe one or more labels specific for DNA. In a further aspect of any oneof the foregoing embodiments, the present invention provides a methodfor sorting the epigenetically modified DNA or chromatin by varying thevoltage, using a sorting laser, varying the magnetic moment, or varyingthe pressure differential across the submicrometer channel. In a furtheraspect of any one of the foregoing embodiments, the present inventionprovides for further analyzing sorted DNA by PCR, nucleic acidhybridization, microarray, ChIP, DNA sequencing, mass spectrometry, andNMR. In a further aspect of any one of the foregoing embodiments, thepresent invention provides for calculating the size of the DNA from asignal magnitude provided by detecting of the one or more labelsspecific for DNA.

The methods may comprise varying the amount of the applied voltage orpressure across the submicrometer channel to sort epigeneticallymodified DNA from DNA that does not provide time coincident detection ofone or more labels specific for DNA and one or more labels specific forone or more epigenetic modifications.

The methods may further comprise using a sorting laser to sort theepigenetically modified DNA from DNA that does not provide timecoincident detection of one or more labels specific for DNA and one ormore labels specific for one or more epigenetic modifications. In someembodiments, the sorted epigenetically modified DNA can be furtheranalyzed by PCR, nucleic acid hybridization analysis, microarrayanalysis, ChIP, or DNA sequencing. The one or more epigeneticmodifications can be selected from methylation of one or more DNAnucleotides, hydroxymethylation of one or more DNA nucleotides,acetylation of one or more histones, methylation of one or morehistones, ubiquination of one or more histones, sumoylation of one ormore histones, phosphorylation of one or more histones, binding of RNApolymerase II, and binding of SWI/SNF.

The methods may further comprise applying a voltage potential orpressure differential across the submicrometer channel, wherein thevoltage potential or pressure differential propels the plurality ofchromatin fragments from the first end of the submicrometer channel toanother end of the submicrometer channel. The method may furthercomprise detecting the label specific for DNA. The method may furthercomprise detecting the label specific for the one or more epigeneticmodifications.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A, 1B and 1C depict exemplary devices of the present invention.FIG. 1A is a photograph of a 100 millimeter diameter wafer containing 27devices, each consisting of a parallel array of 16 submicrometer fluidicchannels for single molecule spectroscopy. Fluidic ports are added (leftside of wafer) to interface with fluid reservoirs leading to channels.The device can be used to collect single molecule time coincident data,in which a voltage gradient can be set up to run chromatin from bottomto top or from top to bottom. FIG. 1B depicts an optical micrograph of15 of the 16 fluidic channels present on one of the 27 devices. FIG. 1Cdepicts a magnified view of one of the fluidic channels. The view is adifferential interference contrast optical micrograph of a typicalnanofluidic channel used in SCAN (single-chromatin analysis at thenanoscale). The narrow region, with a 500 nm wide and 250 nm deepcross-section, was used during fluorescence detection. 432 of thesechannels were assembled on a single 100 mm diameter fused silica wafer.The scale bar is 10 μm.

FIG. 2 depicts a block diagram of one embodiment of the device of thepresent invention. Lasers may be used to excite fluorophores associatedwith chromatin directed by electrokinetic flow through a nanoscaledevice mounted on an imaging platform such as a confocal microscope.Photons emitted by the DNA specific label and the epigeneticmodification specific label may be separately detected usingphotodetectors such as avalanche photodiodes (APD). Alternatively, otherdetection regimes may be employed in the devices and methods of thepresent invention such as detection of magnetic or electricalproperties. Similarly, methods and devices other than those requiring amicroscope may be employed for data collection such as solid statemethods and methods allowing data collection for multiple fluid channelssimultaneously.

FIG. 3 illustrates a microfluidic device of the present inventioncomprising an inspection or detection volume suitable for singlemolecule detection (SMD). Depicted is a nanoscale channel andapproximately 0.18 femtoliter inspection volume through whichfluorescently labeled materials flow. Three classes of fluorescenceemitting particles may be detected. These include DNA label bound to DNAwhich lacks nucleosomes comprising labeled epigenetic markers (darkcircles—labeled with TOTO-3 Fluorophore), nucleosomes comprising labeledepigenetic markers that lack a DNA label (light circles, labeled witheGFP Fluorophore) and chromatin comprising nucleosomes comprisinglabeled epigenetic markers on labeled DNA (dark and light circlecomplexes). Chromatin may be loaded onto the device at a sufficientlylow concentration such that the probability that two separate moleculesoccupy the inspection volume simultaneously is <0.005.

FIGS. 4A and 4B depict fluorescence optical micrographs using fieldillumination aid in visualizing DNA flow through parallel networks offluid channels. FIG. 4A illustrates a micrograph showing snapshot offluorescently-labeled DNA resting inside fluid channels. FIG. 4Billustrates a micrograph (with time lapse) showing the samefluorescently labeled DNA during flow and single molecule isolation(constricted region).

FIGS. 5A and 5B depict the results of a DNA fragment sizing experiment.FIG. 5A depicts a photon burst histogram for lambda phage DNA that hasbeen digested with HindIII. The positions of the peaks depend on thelength of DNA fragments; the peak area is proportional to the relativeconcentration of each fragment. FIG. 5B depicts a plot of the burst sizeas a function of the known fragment size. The dashed line is a linearleast-square fit indicating the ability to accurately determine the sizeof various DNA fragments.

FIGS. 6A and 6B depict single molecule data collected by a device of thepresent invention. FIG. 6A depicts the number of photons detected inseparate spectral channels as a function of time when quantum dots(bottom) bound to organic Alexa Fluor 488 (AF488 top) molecules areelectrokinetically flowed through a microfluidic channel. FIG. 6Bdepicts a small expanded time interval in order to see three instancesof coincident detection where the quantum dot and AF488 label passthrough the focal volume at the same time.

FIGS. 7A and 7B depict single molecule data collected by a device of thepresent invention for detection of fluorophore labeled nucleic aciddendrimers. FIG. 7A shows Photon burst scans reveal the Y-shaped nucleicacid engineered (NAE) structure for a 3R1G (3 Red (dark circles), 1Green (light circle) fluorophore) label. Shaded bands indicate timecoincident detected bursts. FIG. 7B illustrates how examination of theburst intensity (height) histogram may reveal easily distinguishedfluorophore content on 1G1R (1 green and 1 red fluorophore) and 4G4R (4green and 4 red fluorophores) labels.

FIGS. 8A and 8B depict an experimental method for preparing two colorlabeled nucleic acid to confer amplicon product identity. The panel onthe right depicts an experimental time trace showing unpurified PCRreaction content used in detection with both color primers and ampliconproducts detected separately at the single molecule level. Extremedetection sensitivity is demonstrated using a single fluorophore of agiven color attached to each molecule.

FIG. 9A depicts the percent product as a function of PCR cycle number,measured using single molecule spectroscopy in submicrometer fluidicchannels. Light shaded circles and dark shaded squares represent thepercent product as a function of green and red primers, respectively.Error bars correspond to one standard deviation.

FIG. 9B depicts the percent product as a function of PCR cycle number,measured using a conventional gel electrophoresis method.

FIG. 10A illustrate a representative 1 minute time scan of DNA-antibodyconjugates at 1 ng/ul. Quantum dot labeled antibody channel on top,Labeled DNA channel on bottom.

FIG. 10B illustrates an expanded view of the time scan on the left, from24-28 seconds, to clearly demonstrate single molecule bursts. Timecoincident bursts in both detection channels are shaded.

FIG. 11 illustrates single molecule data collected by a device of thepresent invention. The shaded areas highlight time coincident DNA andnucleosome detection which indicates single molecule detection of DNAand nucleosomes simultaneously.

FIG. 12 illustrates time coincidence of GFP tagged histone H2B (H2B-GFP)nucleosomes and TOTO-3 labeled DNA. Single molecule events with eachfluorophore color may be analyzed for their time-coincidence to identifybound DNA-histone complexes, or nucleosomes. Under the conditionstested, over 95% of the coincidence bursts occur within +/−1 time bin(bin time is 500 microseconds), illustrating excellent co-localizationof the DNA and histones. This sample may be prepared using a 5-minmicrococcal nuclease digestion and labeled with TOTO-3 at 1:5 (dye:basepair).

FIG. 13 depicts coincidence statistics from experiments using threedifferent molar ratios of TOTO-3 to DNA, or no TOTO-3. The number ofphoton bursts detected in the red channel (TOTO-3-labeled DNA) and greenchannel (H2B-GFP-tagged nucleosomes) are reported, along with theduration of each bursts and the percentage that are coincident withbursts of the other color. Coefficients of variation are also reported,calculated from the total events for each experiment. Note that whenTOTO-3 is used to label DNA at concentration ratios of 1:10 or higher,greater than 95% of the GFP-containing particles are also carrying theDNA intercalator TOTO-3. This indicates an abundance of flowingchromatin is intact on our devices. The red bursts seen in the absenceof TOTO-3 are due to residual TOTO-3 dye left from prior runs on thereusable devices.

FIGS. 14A, 14B, 14C, and 14D illustrate that dilution analysis confirmsGFP (nucleosome label) and TOTO-3 (DNA label) coincidence measurements.HeLa cells expressing H2B-GFP are admixed with HeLa cells lacking theGFP transgene in the proportions shown on the X-axis, then preparedchromatin from the mixtures using micrococcal nuclease digestion timesof 15 and 5 minutes. The chromatin is labeled with TOTO-3 andcoincidence measurements are performed. FIGS. 14A and 14B show a plot ofthe number of fluorescent events per minute for which TOTO-3 (FIG. 14B)and GFP (FIG. 14A) signals are coincident as a function of theproportion of H2B-GFP expressing Hela cells used to prepare chromatin.FIGS. 14C and 14D show the coincidence rates from the 5 min MNasedigestions are normalized to the number of H2B-GFP fluorescent events(FIG. 14C) and TOTO-3 events (FIG. 14D). The rate of coincidence per GFPevent is constant in all dilutions of GFP-tagged chromatin. The rate ofcoincidence per TOTO-3 event diminishes as the GFP input diminishes.

FIG. 15A and FIG. 15B depicts time coincident detection of methylatedDNA and a reagent designed to detect methylated DNA, and the lack oftime coincident detection of the reagent and unmethylated DNA.

FIGS. 16A, and 16B depict micrographs of embodiments of exemplaryanalytical and preparative devices of the present invention. FIG. 16Ashows a light micrograph of a sorting device in which 10 Kbp DNAfragment labeled with YOYO-1 is run from bottom to top, with the top twobifurcations (from one bifurcation point) representing two possibleoutflow tracts. FIG. 16B shows a long exposure fluorescent micrograph ofYOYO-1 labeled DNA flowing through the sorting device from bottom totop. In this image, the electrode in the left arm of the outflow tractwas charged, directing DNA to the left collection chamber. Note that theelectrode in the right arm can be charged by a high-speed switch that isprogrammed to respond to observations made in the inspection volume,just below the bifurcation. The sample, which could be chromatin, couldbe labeled with TOTO-3 and analyzed and/or sorted similarly.

FIG. 17 shows a schematic diagram an experimental platform including awafer mounted on a confocal fluorescence microscope. Two overlappedlasers illuminated a 1.3 μm length of the nanofluidic channel and formedan inspection volume of 0.16 fL. Collection of the dim, fluorescentsignature for each molecule was achieved using a confocal aperture,which spatially restricted the optical collection to the inspectionvolume, and avalanche photodiodes (APDs), which provided single photondetection.

FIG. 18 shows a process of single molecule detection and two-colorcoincidence analysis. Time-trace record of photon bursts observed byeach APD, showing 0.25 seconds of a 15 minute nanofluidic SCAN. A burstwith a sum of 10 or more photons satisfied a threshold condition and wasdesignated a single molecule detection (SMD) event, shown here by acircle symbol in the top graph or bottom graph identifying DNA andhistone H2B, respectively. Intact chromatin fragments, identified on thegraphs with a vertical shaded bar spanning both graphs, were identifiedby time-coincident detection of both a red and green event.

FIG. 19A and FIG. 19B show an exemplary nanofluidic SCAN of GFP-HeLachromatin at different digestion times. Chromatin was isolated from thenuclei of wild-type HeLa cells and HeLa cells with a H2B-GFP fusiontransgene and then analyzed by SCAN for 15 minutes. (A) A TCHillustrates the absence of coincident two-color SMD events whenanalyzing chromatin from wild-type HeLa nuclei. With GFP-HeLa chromatinfrom the 5 minute digestion, a central Gaussian peak, corresponding tointact chromatin molecules emitting two fluorescent colors, emerged froma background of uncorrelated events. By integrating the area under thepeak and subtracting the uncorrelated background, we observed more than16,000 two-color chromatin molecules. (B) The proportion of two-colorchromatin molecules increased in direct proportion with GFP-HeLa nucleicontent, as described by a linear fit with R²=0.98 and R²=0.95 for the 5and 15 minute digestion assays, respectively. Error bars represent thepropagated error from SMD (single molecule detection) of both the boundand unbound molecules.

FIG. 20 shows detection of DNA methylation. (FIG. 20A) Unmethylated(top) and methylated (bottom) DNA samples labeled with TOTO-3 were bothincubated with a molar excess of MBD1 probes labeled with Alexa Fluor488 and then analyzed for 15 minutes. The emergent peak in the bottompanel demonstrates SMD of methylated DNA. The molar excess of labeledMBD1 contributed to the background of uncorrelated events within eachexperiment. (FIG. 20B) We analyzed mixtures of methylated andunmethylated DNA. The proportion of dual-color labeled MBD-DNA was shownto increase with methylated DNA, as described by a linear fit withR²=0.99. Error bars represent the propagated error from SMD of both thebound and unbound molecules.

FIG. 21 shows a histogram of time duration of TOTO-3 SMD events.

FIG. 22 shows a burst separation graph for separate fluorescent dyecolors.

FIG. 23 is a gel showing chromatin fragments prepared during differentbatches of MNase digestion.

FIG. 24 is a gel showing chromatin fragments prepared during differentbatches of MNase digestion.

FIG. 25 is a gel showing effectiveness of methylation reactionsfollowing DNA digestion with the methylation sensitive restrictionenzyme Hpal.

FIG. 26 is a gel showing that the Alexa Fluor 488 labeled MBD1 retainedits specificity for methylated DNA.

FIG. 27A depicts a channel comprising a plurality of bifurcation points.

FIG. 27B depicts a channel comprising an upstream channel fluidicallyconnected at a branch point to more than two downstream flow paths orchannels.

FIG. 28 depicts a schematic illustration of parallel fluidic channelswith multiple input reservoirs.

FIG. 29 depicts a schematic illustration of parallel fluidic channelswith multiple output reservoirs.

FIG. 30 depicts a schematic illustration of a single input fluidicchannel leading to several possible output fluidic channels.

FIG. 31 depicts a schematic illustration of a fluidic chip interrogatedoptically with a lens for imaging the resulting fluorescent emissionfrom each channel independently on a CCD, CMOS array or other arrayedphotodetector.

FIG. 32 depicts a schematic illustration of a method of fabricating adevice with parallel fluidic channels.

FIG. 33 depicts a scanning electron micrograph of eight parallel fluidicchannels fabricated in a fused silica wafer.

FIG. 34 depicts a schematic illustration of a device having a commoninput and output reservoirs and a central region of 40 parallelnanofluidic channels.

DETAILED DESCRIPTION OF THE INVENTION

Microfluidic and nanofluidic platforms that combine high throughputdetection and analysis of single chromatin fragments can overcome thelimitations of existing epigenomic methods. In some embodiments, thepresent invention includes compositions and methods for flowing anddetecting single native chromatin molecules. In some embodiments,methods for flowing and detecting single native chromatin molecules canbe done by analyzing time-coincident fluorescent signatures of both theDNA and histone proteins within the chromatin at high throughput. Insome embodiments, the invention includes methods to study an epigeneticmark in DNA using conditions that paralleled chromatin studies and afluorescently labeled probe that can bind to methylated DNA. Thesemethods can be referred to as SCAN (single-chromatin analysis at thenanoscale) and are the first demonstration of single-moleculehigh-throughput epigenetic analysis.

In one aspect, the present invention provides methods and devices fordetection of epigenetic modifications at single molecule resolution. Themethods and devices provided herein may be applied to epigeneticanalysis of individual chromatin, or DNA molecules. Single moleculestudies of chromatin allow simultaneous detection of multiple epigeneticmarks on an individual chromatin fragment and require very small amountsof input cell material. This dramatically improves the quality ofepigenetic analyses and opens up new avenues of investigation at thesingle-molecule level. The devices and methods provided herein can beequally well applied to the analysis of other chromatin associatedfactors that might not formally be considered to be epigenetic innature. These methods can provide simultaneous detection of multipleepigenetic marks and may address fundamental questions in developmentalbiology and human health.

Methods

The invention provides for methods for performing epigenetic analysis ofgenetic material. One aspect of the invention provides for a method forperforming epigenetic analysis on a genetic material in a channel,comprising (a) flowing the genetic material through said channel,wherein said genetic material is labeled with a plurality of labels, atleast one of which is specifically complexed with an epigenetic markeron said genetic material, and at least one other label is complexed witha protein and/or nucleotide of said genetic material; (b) illuminatingthe channel to create a plurality of interrogation volumes, each ofwhich is confined by walls of said channel and a beam of light; and (c)detecting the at least one label and the one other label from the sameor distinct interrogation volumes of said plurality to generatetime-correlated resolution of said first and second label, therebyperforming said epigenetic analysis.

In another embodiment of the invention, the detection of the first andsecond label is performed simultaneously. Performing epigenetic analysison a genetic material in a channel can comprise flowing the geneticmaterial through an illuminated interrogation volume, said interrogationvolume being confined by walls of said channel and a beam of light,wherein said genetic material is labeled with a plurality of labels, atleast one of which is specifically complexed with an epigenetic markeron said genetic material, and at least one other label is complexed witha protein and/or nucleotide of said genetic material, and simultaneouslydetecting the first and second label within the interrogation volume,thereby performing said epigenetic analysis.

In some embodiments, a method for producing real-time analysis data on amaterial in a channel can comprise flowing the material through anilluminated interrogation volume, said interrogation volume beingconfined by walls of said channel and a beam of light, wherein saidmaterial is labeled with a plurality of labels, at least one of which isspecifically complexed with a marker on said material, and at least oneother label is complexed with said material, detecting the first andsecond label within the interrogation volume in real-time, therebyproducing said real-time analysis data, and displaying said real-timeanalysis data on a display device, wherein said real-time analysis datacomprises information on said first and second label.

The present invention can increase the sensitivity of epigenomicanalysis, which can allow the use of samples comprising single cellquantities of genomic material. It can also increase the number ofsimultaneous epigenetic analyses possible. The present inventionprovides a method that allows simultaneous analysis of multipleepigenetic markers (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or more) using extremelylimiting amounts of materials, a few orders of magnitude lower than iscurrently needed for histone analysis. In some embodiments, the methodrelies on single molecule three-color fluorescence microscopy imaging ofmore than 4,000 DNA and chromatin fragments per minute flowing through asingle electrophoretic channel on a nanofluidic device. Using thisdevice it is possible to dramatically expand sample throughput and thenumber of epigenetic marks analyzed using parallel networks of fluidicchannels and simultaneous optical analysis. Sorting capabilities mayallow recovery of the imaged and sorted materials for massively parallelDNA sequencing.

In some embodiments, the present invention provides for full redundantcoverage of whole genomes for epigenetic modifications. In some cases,the present invention provides methods and devices for 2×, 3×, 4×, 5×,6×, 7×, 8×, 9×, 10×, or higher coverage of genome (e.g. mouse, human,vertebrate, animal, plant, fungal, archaeal, bacterial etc.) in our fullanalysis. In some cases, 1,000; 2,000; 3,000; 4,000; 5,000; 6,000;10,000; 15,000; 20,000; 30,000; 50,000; 75,000; 100,000 or more DNAfragments per minute with an average target fragment size of 1,000;2,000; 3,000; 4,000; 5,000; 6,000; 10,000; 15,000; 20,000; 30,000;50,000; 75,000; 100,000 bp or more, provides 2× or higher genomecoverage in fewer than 3 hours, 2 hours, 90 minutes, 75 minutes, 60minutes, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 3minutes, 2 minutes, 1 minute, 30 seconds, 15 seconds, 10 seconds, 5seconds, or less. The time to analyze data may increase as the number ofchromatin marks detected increases, but this does not limit the speed ofdata acquisition. The device architecture may be modified to allowsample sorting and recovery for sequencing. This embodiment can takeadvantage of real-time data analysis and control of switching circuitry.The analytical speed may depend on software design, computer speed andthe specific circuitry chosen. It is worth noting that in commercialflow cytometry equipment like the BD-Biosciences FACS Aria, sort ratesof 30,000 events per second are routine. The devices of the presentinvention can be capable of comparable or even faster sorting rates. Forexamples, the devices of the invention can be capable of sorting aboutor more than about 30,000, 35,000, 40,000, 45,000, 50,000, 55,000,60,000, 70,000, 80,000, 90,000, 100,000 events per second or more. At30,000 imaging events per second of 20,000 bp fragments, the entiremammalian genome can be analyzed in 5 seconds.

In some embodiments of the invention, the genetic material can becharacterized in less than about 1, 0.5, 0.1, 0.01, 0.001, or 0.0001seconds. The characterization can be performed by a processor thatreceives signal corresponding to properties of the genetic material froma detection module. In one embodiment, the detection module is anavalanche photodiode. The detection module can transmit signalcorresponding to more than one or a single property or marker. Thedetection module can transmit signal corresponding to more than twoproperties or markers. The processor can determine the presence orabsence of an epigenetic marker based on said signal.

The invention also provides for methods for performing analyticaltechniques that are analogous to precipitation assays in asingle-molecule format. For example, any immunoprecipitation assay canbe improved using the methods, systems, and devices of the invention toobtain single-molecule resolution and/or effect high-speedcharacterization of a molecule or object.

Sample

The methods and devices of the present invention may be suitable for theanalysis of samples comprising an object, a genetic material, a nucleicacid (e.g., DNA, RNA, or any hybrid thereof), a peptide, a protein, anaptamers, any fragment thereof, or any combination thereof. Any of theseobjects may be single objects, e.g., a single nucleic acid or a singlechromatin.

The term “objects” can refer to any molecule, single molecule, complex,cell, cellular component, or bead described herein. For example, thesystems, devices, and methods may be used to sort and/or analyzebiomolecules including but not limited to genetic material, nucleicacids (e.g. DNA, RNA, and hybrids thereof), nucleic acid fragments,proteins, protein fragments, aptamers, carbohydrates, lipids, nucleicacid-protein complexes, protein-protein complexes, and any combinationthereof. The object can also be a cell, cellular component, or cellfragment. For example, the systems, devices, and methods may be used tosort and/or analyze other molecules, including polymers, organicmolecules, small organic molecules, drugs, drug targets, and compounds.The subject device may also sort and/or analyze particles, such asbeads, vesicles, and lipid vesicles. In some cases, the systems,devices, and methods provided herein may be utilized to sort and/oranalyze nucleic acids that have specific bound proteins or alteredchemical states for epigenetic analysis. In some cases, the inventionprovides for sorting and/or analysis of chromatin, which encompasseswhole chromatin and chromatin fragments, and/or histones. The object tobe sorted and/or analyze can be of a variety of sizes. For example, theobject to be sorted typically has a dimension that is less than thesize, diameter, or width of a channel in the sorting system. In someembodiments, all dimensions of the object are less than the width andheight of the channel. In other embodiments, the length of the objectmay be greater than the width of the channel, e.g., an elongated nucleicacid. As described herein, the objects to be sorted can be measured forintrinsic properties, or may be labeled with another molecule thatcomplexes with the object. Examples of such labels include fluorescentdyes, e.g., a quantum dot that is optionally conjugated to a nucleicacid probe. Other labels that can be utilized include intercalatingdyes, e.g., YOYO-1, TOTO-3, Syber Green, and ethidium bromide.

The genetic material can be a single molecule, or a complex formed byindividual molecules. In some embodiments, the genetic material is achromatin (encompassing chromatin fragments) or a single chromatin(encompassing single chromatin fragments). The chromatin can be analyzedfor the presence or absence of an epigenetic marker, or a number ofepigenetic markers present on the chromatin.

The sample may be obtained from any cell or tissue source. In somecases, the methods and devices of the present invention may be suitablefor analysis of samples comprising a small or limiting amount of DNAincluding but not limited to: MEF DNA, ES DNA, Dnmt1−/−ES DNA, Mouseblastocyst DNA, DNA from a microdissected tumor, DNA from apre-implantation embryo, MEF chromatin, ES chromatin, Dnmt1−/−chromatin,and blastocyst chromatin.

The object, genetic material, nucleic acid, DNA, chromatin peptide,protein, or any combination thereof may be fragmented by any means knownin the art prior to (or after) flowing it into a channel, and differentapproaches may be preferred depending upon the source of the DNA orgenetic material. For abundant and concentrated sources totaling severalmicrograms in 200 μl or a larger volume, a standard sonicator probe maybe inserted into the tube containing the DNA. An alternative is to useDNAseI to fragment DNA. Another alternative is to use micrococcalnuclease or any other suitable nuclease such as a double stranded DNAexo or endo nuclease to fragment chromatin.

The methods provided herein are useful for analyzing methylation statesin DNA or other genetic materials taken from extremely low abundancesources. A single mammalian cell can hold approximately 3 pg of genomicDNA and one use of the present invention can be to analyze DNA fromapproximately 30 laser microdissected tumor cells, single mouse morulaethat contain eight cells and blastocysts that contain approximately 40cells.

When working with small quantities of cells or any other samplesdescribed herein, purification methods that use proteinase treatment andorganic extractions followed by alcohol precipitation can be quitereliable, and in some cases they require glycogen or tRNA as a carrierto maximize DNA recovery. An alternate approach for solubilizing DNA isto disrupt the cells physically, by placing a tube containing them in acup horn sonicator. Sonication of microdissected cells and embryos mayfree DNA from nuclei, avoid sample loss during purification and make itunnecessary to use carriers. These crude extracts may be compatible,under the proper buffer conditions, with nanoscale flow and antibodydetection. Sonication may produce debris that may clog the nanoscalechannels of a device of the present invention, unless debris is belowthe cross sectional channel dimensions in the device, which may measure,e.g. approximately 500 nm×500 nm. Centrifugation may be sufficient toremove particles. Alternatively devices in which a grid, whose spacingis smaller than the channel opening, precedes the input channel may beprepared—this may prevent channel clogging by filtering particulates onthe device before analysis. Also, with sonicated materials, smallamounts of EDTA and SDS may be needed to preserve DNA integrity andsolubilize cellular materials. When sonicating pre-implantation embryos,it may be necessary to remove the zona pellucidae using acid Tyrode'ssolution. The zona pellucida is a tough proteoglycan layer that mayresist sonication.

The genetic material that can be analyzed by the subject method caninclude pre-implantation embryos. For pre-implantation mouse embryos,the zona pellucida may be removed using acid Tyrode's solution beforecross-linking chromatin with formaldehyde. The proteoglycan rich zonamight interfere with formaldehyde access to the nucleus. Sonication maybe performed using a cup horn sonicator containing a tube holding asingle embryo. Specific epigenetic marks may be detected, one at a timeor in combination.

Laser microdissected materials may also be analyzed by the methods anddevices of the present invention. Analysis of chromatin inmicrodissected samples may be performed using frozen sections. By usingsections from frozen tissues, the cross-linking time may be controlledthe cell-to-cell differences in exposure to formaldehyde may be limited.Paraffin embedded samples may also be analyzed.

Both native chromatin as well as preserved chromatin prepared bycross-linking proteins to DNA using formaldehyde can be used in thesubject analysis. Cross-linking has the advantage of fixing andstabilizing proteins at their points of contact on the DNA, and has beenwidely used in ChIP, ChIP-Chip and ChIP-Seq studies. Where deemed,cross-linked chromatin from ES and MEF cells can be used. The chromatinmay be stained with QD-labeled antibodies recognizing differentepigenetic marks including DNA methylation, DNA hydroxymethylation,bound RNA pol II, modified or unmodified histones, or any other knownepigenetic mark, then individual molecules may be imaged in a nanoscaleflow channel of the present invention during voltage-directed flow. Bycombining different antibodies, each labeled with a different QD whosespectral properties are distinguishable, and the coincidence, or mutualexclusion, of each feature may be studied. Multiple independent featuresmay be analyzed simultaneously (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 14, 15, 16, 18, 20, 25, 30 or more).

Input materials, including genetic materials, DNAs, chromatins, or anyother sample to be analyzed may be prepared by any number of methodsknown in the art such as using proteinase K treatment, phenachloroformextraction and ethanol precipitation. Similarly, chromatin may beprepared by any number of methods known in the art including native ornon-native extraction methods. An important variable that may influencehow many kilobases of sequence to be analyzed per unit time is theaverage length of the DNA fragments analyzed. The magnitude of photonbursts from YOYO-1-labeled DNA fragments from 600 bp to 27,000 bp can bereadily analyzed and sized. Accordingly a wide range of fragment sizescan be used. By using large fragments, total genome analysis may beprovided by querying fewer fragments and photon burst events. However,using large fragments provides a limited ability to resolve where theDNA methylation is located. In some cases, comprehensive bisulfate basedanalysis of DNA fragments preliminarily sorted on the basis of anti5-methylcytidine antibody binding may be performed by the methods of thepresent invention.

In some cases, the methods of the present invention utilize chromatinthat has already been subjected to a ChIP procedure to enrich the inputmaterial carrying the marks of interest. For example, a ChIP may beperformed with anti-H3K4me3 and this material may be used in a nanoscaledevice of the present invention to detect H3K27me3. This enriched inputmaterial can then be used to optimize buffer conditions for epigeneticmark detection on the device. It is worth noting that with extremelyhigh surface area to volume ratios present in the nanoscale channel,buffer conditions are critical to minimize surface-driven artifacts aswell as to ensure proper antibody to antigen interactions.

Labels

The object or genetic material in the sample can be complexed,pretreated, or mixed with one or more labels. In one embodiment, thegenetic material is labeled with a plurality of labels, at least one ofwhich is specifically complexed with an epigenetic marker on saidgenetic material and at least one other label that is complexed with aprotein and/or nucleotide of said genetic material. Labels includefluorescent dyes, quantum dots, magnetic particles, metallic particles,and colored dyes. Examples of dyes are described herein. The dyes orlabels can be conjugated to binding moieties such as antibodies, nucleicacids, proteins, aptamers, affinity clamps, peptides, naturallyoccurring proteins and protein domains that bind to target proteins ofinterest. The binding moieties can be specific or generic. In someembodiments, one binding moiety is specific to an epigenetic marker anda second binding moiety generically binds to nucleic acids, proteins, orbiological molecules.

The genetic materials to be analyzed can be analyzed with or without alabel. In some embodiments, the genetic material is not labeled anddetection techniques not reliant on labels can be used to characterizethe genetic material. For example, electrical conductance and UVabsorbance can detect DNA in the absence of a label. In otherembodiments, the genetic material to be analyzed is labeled such thatproperties of the genetic material can be observed. Labels can bespecific to certain trains of the genetic material, or the labels can begeneric. Generic labels include labels that bind non-specifically tonucleic acids (e.g., intercalating dyes, nucleic acid groove bindingdyes, and minor groove binders) or proteins. Examples of intercalatingdyes include YOYO-1, TOTO-3, Syber Green, and ethidium bromide. In somecases, the method provides alternative approaches to label chromatin,other than YOYO-1 or TOTO-3.

Samples may be labeled with specific labels. Specific labels can includedetectable moieties (e.g., dyes, metal particles, radioactive particles,and magnetic particles) that are conjugated to moieties that bind tospecific genetic markers. Chromatin may be labeled using such specificlabels. Chromatin isolated from embryos at different stages fromfertilization to gastrulation can be labeled with a QD-labeled antibodyrecognizing H3K27me3, H3K4me3 and 5-methylcytidine, or any other knownepigenetic marker using a different color QD for each antibody.Alternatively, any suitable labeling reagent may be used to labelepigenetic modifications such as a binding agent that specificallyrecognizes epigenetic markers as provided herein including but notlimited to labeled antibodies, antibody fragments, minibodies,affibodies, avimers, aptamers, other proteins that bind epigeneticmarkers or groups of markers associated with chromatin such as MDB1.Labeled binding agents used herein can complex with any target ofinterest described herein. Targets of interest can include MBD1, RNA PolII, RNA, DNA, SWI/SNF, mRNA, pre-mRNA, miRNA, piRNA, lincRNA, and siRNA.Similarly, suitable labels may include but are not limited to QD,organic fluorophores, or agents that can be detected by changes inmagnetic or electrical properties. Where desired, the labeled chromatinmay be sorted on a device of the present invention and fractions witheach combination of these marks may be isolated for subsequent highthroughput (e.g. Solexa, Illumina, 454/Roche, etc.) sequencing.

Samples may be labeled with a label conjugated to a binding moiety thatcomplexes with a target. In some embodiments, the target is a specificDNA sequence. Specific DNA sequences can be recognized using labeledbinding moieties, such as labeled probes, nucleic acid sequences, andDNA binding proteins. DNA binding proteins include, but are not limitedto, those described in Jamieson, Nat Rev Drug Discov 2: 361-8 (2003),Urnov, Nature 435: 646-51 (2005), Moscou, Science 326: 1501 (2009), andNielsen Science 254, 1497 (1991).

In some instances, two generic dyes and one specific dye correspondingto a first property can be used to label a sample. This can allow fordistinction between free dye, objects without the first property, andobjects with the first property. In other embodiments, two generic dyesand two specific dyes may be used to label a sample, where the firstspecific dye corresponds to a first property and the second specific dyecorresponds to a second property. In addition to detecting free dye,objects without a first property, and objects with a first property,this would allow for detection of objects without either the first orsecond property, objects with the second specific property, objectswithout the second property, and objects with both the first and secondproperty.

Nucleic acid binding agents can be used to label a sample. Non-limitingexamples of labels or labeling moieties include probes of a specificsequence, intercalating dyes, and minor groove binders. Generally,fluorescent intercalators can be dyes that bind to double-stranded DNAor double-stranded RNA by inserting themselves in between a neighboringbase pair. Generally, minor groove-binders can be dyes that bind to theminor groove of double-stranded DNA. There are still other dyes that maybind to nucleic acids via multiple modes, including electrostaticinteraction between a positively charged dye and the negatively chargednucleic acid. In some cases, it is desirable to image all chromatinfragments, regardless of their epigenetic states. For example, one mayidentify the proportion of sites in the blastocyst genome that carry theH3K27me3 and H3K4me3 epigenetic marks. At least two alternate approachesare available for labeling all chromatin fragments, independent ofintercalating dyes. One approach is to label Alexa-coupled nucleotidesto the 3′ end of each chromatin fragment using terminal deoxynucleotidetransferase (TdT). The Alexa fluor chosen may be spectrally distinctfrom the QDs chosen for the antibodies. This TdT-mediated labeling isnot limited by the cross-linking and only depends on a 3′-OH group atthe end of each chromatin fragment. Another approach is to label ahistone rather than the DNA using antibody recognizing H1 (anti-H1) orone of the core histones. With 80% of the genomic sequences associatedwith nucleosomes only extremely rare chromatin fragments will remainunlabeled by anti-H1 in preparations of chromatin fragments in the20,000 bp size range.

Samples can be labeled with QD-anti-methyl-C for detection of methylatedDNA. In some cases, methylcytosines directed towards the nucleosome maybe inaccessible to QD-anti-methyl-C in cross-linked chromatin. This canprovide lower QD fluorescence emissions for a chromatin fragmentrelative to the same DNA sequence lacking cross-linked proteins.However, linker DNA between nucleosomes may be unaffected by crosslinking, so linker DNA is as accessible in purified DNA as in crosslinked chromatin. Second, even for DNA wrapped around nucleosomes, lessthan half of the signal may be lost because methylcytosines extend intothe major groove of DNA so access of antibody in solution to the majorgroove is what determines QD-anti-methyl-C binding to methylated DNA;DNA is wrapped around the external surface of nucleosomes with less thanhalf the major groove surface area in sufficiently close proximity tothe histones to exclude the antibody; thus most of the methylcytosinesin a nucleosome is accessible to QD-anti-methyl-C. The combined accessof methylcytosines in nucleosome-associated DNA and linker DNA to thesolution phase may provide adequate signals for sequences containingmethylated DNA.

In some cases, methylated DNA in chromatin may be detected by labelingsamples with QD-anti-methyl-C that can bind to methylcytosines andQD-anti-H1 or a 5′ or 3′ end label that can bind to chromatin. This canbe used, for example, to label chromatin from cultured ES cells toanalyze the chromatin for methylation. Dnmt1 mutant cells may beutilized as an excellent negative control to assess the specificity ofthe antibody binding to methylcytosine in the context of chromatin. Inthis case, chromatin can be from wild type and Dnmt1 mutant ES cells,which may be prepared, similar as to in ChIP, using the above-mentionedlabels. The methylcytosines labeled using QD-anti-methyl-C and thelabeled chromatin may be analyzed on the nanoscale device of the presentinvention. This method may identify the proportion of fragments bearingmethylcytosine marks and the density of those marks in chromatin. Acomparison can be made with the data obtained using purified DNA. Insome cases, ES and MEF cell chromatin may be labeled using QD-anti-H3K4me3 and QD-anti-H3K27me3.

In some embodiments, three labels are mixed with the sample. In anexemplary embodiment, the genetic material can be ES and MEF chromatin.A third QD-labeled antibody may be added to the analysis to detect RNApol II. By combining this antibody with QD-anti-H3K4me3 andQD-anti-H3K27me3, the methods provides for correlating the epigeneticmark placement with a marker for gene expression competency. Inaddition, the genome may be queried for coincidence of DNA methylationand H3K27me3.

In some embodiments, the sample is processed by the system withoutremoval of free dye and/or free label. The free dye and/or free labelcan be characterized appropriately by the system by use oftime-correlated or simultaneous detection of a plurality of properties.In other embodiments of the invention, free dye and/or free label isremoved from the sample prior to sorting. The concentration of the freedye and/or free label in the sample can be about, less than about, orgreater than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 30, 50 times the concentration ofthe dye and/or label that is complexed with the object or geneticmaterial.

Fluid Movement

The sample, which may contain a genetic material of interest that hasbeen labeled with one, two, three, four, or more labels, can be loadedinto a device of the invention for detection and analysis. The samplecan be loaded to an input reservoir on the device. The sample can form acontinuous body of liquid as it moves through the device. Fluid carryingthe genetic material, or the genetic material alone can be directedthrough the channels using an external pressure source, an internalpressure source, electrokinetics, magnetics, or some combinationthereof. The external or internal pressure source can be a pump, e.g., aperistaltic pump, syringe pump, or a pneumatic valve pump. The flow ofthe genetic material and/or the flow of a solution carrying the geneticmaterial can be reversed. The reversal can be effected by reversingpolarity of a voltage gradient and/or reversing a pumping action.

A sample, or a plurality of samples, can be loaded into one or aplurality of channels. For example, the sample or plurality of samplescan be loaded into about, up to about, or less than about 1, 2, 5, 10,50, 100, 500, 1000, 5,000, or 10,000 channels. The loading and/oranalysis of sample can be sequential or simultaneous. The sample may beloaded into a common input reservoir or individual reservoirs. Thechannels can be arranged in a variety of configurations, includingparallel channels, radial channels, branched channels or a combinationthereof.

Flow of the chromatin or any other sample type described herein throughnanofluidic devices may be induced electrokinetically using voltagesapplied to reservoirs of the device. The flow rate inside these devicescan be controlled over a wide range with exquisite accuracy. The flowrate can be controlled to an accuracy of about, or better than about0.00001, 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,5, 10, 50, 100, 500, 1000, 5000, or 10,000 fL/s or μL/s. The flow ratecan be about, less than about, or greater than about 0.00001, 0.00005,0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100,500, 1000, 5000, or 10,000 fL/s or μL/s. Molecules can be driven atrates of greater than about several hundred or thousand per second ineach channel independently. This provides not only the opportunity toincrease the rates of DNA and chromatin throughput, but it will allowthe use of an electrical method for rapid single-molecule sorting. AY-shaped separation chamber may be placed immediately following thedetection region, the proverbial “fork in the road.” In some cases, amore highly branched separation chamber may be utilized in parallel orin series to achieve a greater number of outflow tracts or collectionchambers. Flow rate can be increased and directed preferentially to onebranch or the other of the fork by briefly increasing the voltage biasin the corresponding part of the fork. In this way, the molecule issorted toward the desired path for eventual recapture. This method ofcontrol is simple and can be applied to either DNA or chromatinfragments, bound to antibody-linked fluorophores, which report theepigenetic state.

In other embodiments of the invention, the flow of the object can bereversed. This can allow for more than one measurement to be taken on anobject within a single interrogation volume. Alternatively, multipleinterrogation volumes can be placed in a channel prior to a branch pointsuch that multiple measurements of the same properties are performed onthe object prior to a sorting or characterization event. For example,two, three, four, five, six, seven, or more interrogation volumes may bepositioned upstream to a branch point, thereby allowing for a pluralityof measurements of the same two, three, four, five, or more propertiesused to determine how to sort or characterize the object.

Illumination and Detection

In one aspect, the present invention provides methods for detectingproperties of a genetic material to perform epigenetic analysis. Theproperties can be detected using one or more labels. The labels can bedetected in one or more interrogation zones. The interrogation zones canbe the same or distinct interrogation zones. In some embodiments,chromatin and/or individual molecules may be interrogated,simultaneously, for DNA methylation and histone modifications.

In some embodiments, the present invention provides methods forsimultaneous or time-correlated detection of various molecularproperties including, but not limited to, fluorescence, such as quantumdot fluorescence signatures. As described herein, the properties of theobject can be measured using a variety of detection techniques. Thedetection step can measure optical, electrical, radioactive, physical(e.g., size, density, thermal conductivity, elasticity, viscosity, andstrength), and/or magnetic properties. The fluorescence signatures canbe observed in real-time and in fact be used as triggering events tocontrol a sorting method as provided herein.

The detection step can include interrogating or inspecting an objectwithin a defined volume. The defined volume can be referred to as aninterrogation volume, inspection volume, or detection volume. In someembodiments, the interrogation volume is an optical volume when opticalsignals are detected. The detection step can include measuring one, two,three, four, five, six, or more properties or signals from the object.For example, an object may have distinct measurable properties thatchange depending on the state of the object. These measurable propertiescan be intrinsic to the object, or can conferred by one or more labelsthat are complexed with the object. Examples of labels that can becomplexed with the object include fluorescently labeled antibodies thatcomplex with epigenetic markers on a nucleic-acid containing chromatin.

The interrogation volumes can be created by illuminating a channel withone or more light beams. The light beams can be from one, two, three,four, or more light sources. The one or more light sources can emit oneor more beams of light that illuminate one or more regions or volumeswithin the one or more channels. The beam of light may be focused by anoptical component, e.g., a high numerical aperture objective. The beamsof light illuminating the channels can create one or more inspectionvolumes that are defined by the walls of a channel and the beams oflight. The dimensions of the beam of light and the channel can definethe size of the inspection volume, as shown in FIG. 3. The inspectionvolumes can have a volume that is about, up to about, or greater thanabout 0.01, 0.05, 0.1, 0.2, 0.5, 0.75, 1, 5, 10, 25, 50, 75, or 100femtoliters.

The detection of properties of the object or genetic material can beperformed in a simultaneous or time-correlated fashion. Simultaneousdetection can occur by measuring two or more properties of the object atone instant in time. For example, two wavelengths of light correspondingto two distinct labels can be measured in a single interrogation volume,or overlapping interrogation volumes. Alternatively, two or moreproperties of the object can be measured at distinct times. This may beperformed if the location of the object within the channel as it istravelling down the channel can be known, predicted, or estimated. Forexample, a first property can be measured at a first interrogationvolume located upstream to a second interrogation volume, where a secondproperty of the object is measured. Correlation of the signals from thefirst interrogation volume and the second interrogation volume can bebased on the velocity of the object through the channel and the distancebetween the two channels. In this manner, 2, 3, 4, 5, 6, 7, 8, 9 or moreproperties of the object can be measured simultaneously or in atime-correlated fashion.

Signal from the one or plurality of channels can be recorded andanalyzed simultaneously and/or sequentially. This can allow for thesimultaneous and/or sequential detection of properties in a plurality ofsamples loaded simultaneously and/or sequentially to a plurality ofchannels. Simultaneous detection combined with the flow of samplethrough channels at high rates allows for thousands of molecules to beinterrogated each second in each of several thousand channels.

The signals measured may have a signal-to-noise ratio of about orgreater than about 1, 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500, 1000,5000, or 10,000. The signal-to-noise ratio can be determined based onthe resolution and/or accuracy of the detector, the light source, thesize of the inspection volume, the power and/or quality of the lightsource, the amount of light used to illuminate the inspection volume,the label used to indicate a property of the genetic material, or anycombination thereof. The signal to noise ratio can be determined by thesignal above the mean photon noise. A statistically significant signalcan be a signal that is at least about 3 standard deviations above themean photon noise.

Sorting

The sorting module of the present invention allows for the detection andreal-time evaluation of objects and collection of the objects. Sortingcan be performed as described in U.S. Provisional Application No.61/231,979, filed Aug. 6, 2009, U.S. Provisional Application No.61/307,827, filed Feb. 24, 2010, U.S. Provisional Application No.61/231,963, filed Aug. 6, 2009, and U.S. Provisional Application No.61/359,266, filed Jun. 28, 2010, and the co-pending case underPCT/US10/44810, filed herewith, each of which is incorporated herein byreference in its entirety. The sorting of the object can be based onone, two, three, four, five, or more properties measured duringdetection. The properties can be measured simultaneous, or in atime-correlated fashion. A processor can be used to interpret the datacollected on the object and determine a desired flow path for theobject.

The sorting can allow for the detection and real-time evaluation ofspectral or other single molecule signatures and the separation andcollection of these specific molecules. In some embodiments, the devicesand methods of the present invention provide on-the-fly evaluation ofboth time and spectrally coincident signatures examined within thehighly confined structures of the device (fluidic channels, nanopores,or otherwise). Single molecule separation using these coincidentsignatures may provide salient features, such as the ability to separatespecific sequences of nucleic acids bound with proteins or separatemolecules in the presence of other biological species or spuriousfluorescence contamination, all with an extremely low rate offalse-positive detection.

One example of such an implementation is to identify rare epigeneticmodifications to histone proteins bound in chromatin. The methods anddevices of the present invention provide unambiguous identification ofthe sequences moving through the channels and provides for selection ofthese molecules through separation, even in the presence of othercellular debris and proteins which often accompany the chromatinstrands.

In other embodiments, the present invention may provide for the recoveryof sorted chromatin. This sorting and recovery may provide fordetermination with nucleotide sequence specificity, the regions of thegenome harboring complex epigenetic profiles of interest in extremelylow numbers of cells by massively parallel DNA sequencing.

In some cases, the color signatures provided by analysis of the labeledchromatin can be observed in real-time and used as triggering events tocontrol a sorting method. Conventional sorting methods often involvevalves and gates that move at the 10's of millisecond time scale orslower. In the present invention, a high-throughput counting in excessof 2,000 molecules/min has been demonstrated and this can be increasedmuch further. Therefore, the present invention allows for a high-speedsorting method. Two high-speed methods of sorting may be used to exertcontrol over a molecule's trajectory, one electrical and one optical.These sorting methods may be implemented in nanofluidic channels using abranched (e.g. Y-shaped) separation chamber for sorting. Additionalbranch points may be incorporated, either in series or in parallel,further increasing the sorting capabilities if needed.

In some embodiments of the present invention, electrical sorting is usedto sort molecules based on detection of events or molecular properties.The present invention provides a device that can rapidly switch thevoltage applied to a branched fluid channel to separate the molecules ofinterest into a collection volume. Appropriate design of the fluidsystem, electronic switching allows for rapidly interrogating largenumbers of molecules in the sorting system.

The present invention also provides for the use of an optical techniquefor particle manipulation involving the use of an intense laser beam tohold or “tweeze” the particles. The intense laser can be used in thepresent invention for sorting molecules. Using an intense infraredlaser, to minimize photodamage to the biopolymers, and high numericalaperture optics, to create a tightly confined optical potential well, aQD-chromatin conjugate may be captured. This technique may be appliedinside the branched separation chamber of the present device and thenthe laser position may be deflected to control the particle trajectoryfor sorting. The optical sorting technique can be performed at a singlemolecule level in a nanofluidic device by utilizing the dielectricproperties of the fluorophore, particle, or quantum dot. In some cases,this sorting technique may be performed using a constant applied voltagefor flow of molecules in a defined direction past the laser. Thissorting technique provides ultra-fast sorting—with speeds that can belimited only by fluid forces, not the sorting laser deflection. Forcesin the optical potential trap are routinely on the order of 10-100 pNand, in this case, can be applied transverse to the flow direction. Flowvelocities in our nanofluidics, under high-throughput conditions,nominally impart a Stokes drag force of approximately 10 pN on a 20 nmdiameter particle. This indicates that deflection of the QD in thepresence of flow is possible, particularly since deflection into theproper direction requires only a fraction of the possible trappingforce. Various methods for high-speed laser deflection and steering arecommonly used in the field of optically phased-arrays, with deflectionspeeds that can extend into the Megahertz range, orders of magnitudefaster than the Kilohertz frequency of single molecule counting events.Our method allows for ultra-fast sorting control at rates approachingthe fundamental limit inside a fluidic structure.

The branched separation chamber with either the electrical or opticalsorting method provides an unprecedented level of purity and speed inthe recovery of specific chromatin sequences. The triggering event forthis sorting method is based upon the QD color signature generatedduring excitation in the laser inspection volume. The emittedfluorescence is observed using high-speed, ultra-sensitivephotodetectors such as avalanche photodiodes (APDs), which outputdigital signals representing the number of photons observed. To initiatea real-time sorting trigger signal, a programmable high-speed hardwareunit can perform the rapid decision making process. A field programmablegate array (FPGA) or other logic device may incorporate severaloperations to make the final sorting decision. The input signalrepresenting fluorescence may be collected using an integrator operationand then buffered to a comparator to decide if a single molecule eventhas occurred. This operation may be performed for each fluorescencecolor and corresponding photodetector, simultaneously. When theuser-specified condition for sorting is satisfied, the sorting triggeris output to either the adjustable voltage supply (electrical sortingmethod) or to the phased array that deflects the infrared laser (opticalsorting method).

Devices

The present invention provides for systems and devices for analyzinggenetic material. In some cases, the systems and devices can be used forperforming epigenetic analysis on chromatin. A subject system typicallycomprises the following components: a channel being adapted to hold anobject in a continuous liquid body in said channel, one or more lightsources configured to illuminate the channel to create one or moreinterrogation volumes, and a detection module configured to detect atleast two types of signals indicative of two distinct properties of theobject in the one or more interrogation volumes. The object can be achromatin, or a single nucleic acid molecule.

In one aspect, the invention provides for a system for characterizing asingle nucleic acid molecule or a chromatin that can comprise a channelconfigured to hold said molecule, one or more light sources configuredto illuminate the channel to create one or more interrogation volumes, adetection system configured to detect at least two types of signalsindicative of two distinct properties of said nucleic acid molecule fromthe one or more interrogation volumes, and a processor programmed toprovide real-time time-correlated resolution of the at least two typesof signals from said one or more interrogation volumes, therebycharacterizing said single nucleic acid molecule.

The invention also provides for a system for characterizing an objectthat can comprise a channel configured to hold said object, one or morelight sources configured to illuminate the channel to create one or moreinterrogation volumes, a detection system configured to detect at leasttwo types of signals indicative of two distinct properties of saidobject from the one or more interrogation volumes, a processorprogrammed to provide real-time time-correlated resolution of the atleast two types of signals from said one or more interrogation volumes,thereby characterizing said object, and a user interface configured toreceive data from the processor and display the data, wherein the datacomprises information on the at least two types of signals from said oneor more interrogation volumes.

The present invention provides for methods and devices for highthroughput multiplexed single molecule analysis and sorting using aplurality of fluidic channels. The channels can be arranged in a varietyof formats, including parallel channels, radial channels, and/orbranched channels. For example, FIGS. 1B, 4A, 4B, 28, and 34 show aplurality of channels arranged in a parallel format and FIGS. 27A and27B show channels arranged in a branched format.

In some embodiments, the systems and devices can also include adownstream sorting module. The devices, which may be nanofluidic ormicrofluidic devices, provided herein may include a Y-shaped branchpoint in the flow channel with the ability to sort materials intoreservoirs at either of the two ends of the branches. In someembodiments, the nanofluidic devices provided herein may includemultiple (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) branchpoints in a parallel or serial fashion which multiple branch points mayfurther provide multiple outflow tracts or collection chambers. Sortingmolecules bearing specific epigenetic marks into separate collectionchambers may allow for their recovery and sequencing. In some cases anentire genome may be sorted in less than 10 hours, 8 hours, 7 hours, 6hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30minutes, 20 minutes, 10 minutes, 5 minutes or less. The devices of thepresent invention are quite stable and data may be collected on a devicethat has been reused many times (e.g. 1, 2, 3, 4, 5, 6, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, 1000, 10000 times or more).

Channels

The devices of the invention can comprise one or more channels or flowpaths. The channels can be fabricated using a variety of techniques,including microfabrication and nanofabrication techniques. The channelscan be made from a variety of substrates, including, but not limited to,silica, mirror polished fused silica, silicon, quartz, glass, orpolymeric materials (e.g., PDMS, plastics.). Channels may be etched,ablated, molded, into the substrate. The channels or flow paths may becoated. The coating can alter the properties of the channels and may bepatterned. For example, the coating may be hydrophobic, hydrophilic,magnetic, paramagnetic, conductive, or be functionalizable depending onthe objects to be sorted. The coating or a material complexed,conjugated, or bonded to the coating may exhibit affinity to one or moretypes of objects. The coating, or a material bound to the coating mayreduce the adherence of an object to the channel. An example of acoating material includes PTFE. The channels may have a cross-sectionthat is shaped like a circle, oval, rectangle, square, trapezoid,triangle, pentagon, or any other shape. The channel may have one or morecross-sectional dimensions, e.g., diameter, width and/or height, that isup to about, less than about, or about 10, 20, 30, 40, 60, 80, 100, 200,250, 400, 500, 550, 600, 700, 800, 900, 1000, 1250, 1500, or 3000nanometers. The dimension of the channel can be selected to be up toabout, more than about, or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 50, 75, or 100 times the width of an object to besorted.

The one or more channels can be fluidically connected to one another atone or more branch points. The one or more channels allow for thesorting of objects in a continuous body of liquid or in a reversiblefashion (as described herein). The branch points may be bifurcations,where a single upstream channel is connected to two downstream flowpaths or channels at a bifurcation point. As shown in FIG. 27A, asorting device can have a plurality of bifurcation points. In otherembodiments of the invention, one or more channels can be fluidicallyconnected at a branch point to more than two downstream flow paths orchannels. As shown in FIG. 27B, a sorting device can comprise anupstream channel fluidically connected at a branch point to fourdownstream flow paths or channels. In some embodiments of the invention,a device has a plurality of branch points, each branch point having two,three, four, five, or more downstream flow paths or channels. The branchpoints may have the same or different number of downstream flow paths.The branch points may be T-shaped, Y-shaped, or any variation thereof.The channels may be straight or curved. The channels can be positionedin two or three dimensions, such that all channels are in the sameplane, or some channels are in different planes.

The methods and devices can include an array of parallel fluidicchannels, numbering between one and several thousand, where each channelhas a width and depth less than one micron and a length of a fewmillimeters. Alternatively, the length of a channel can be about, up toabout, greater than about, or less than about 0.01, 0.1, 1, 10, 100,1,000, or 10,000 millimeters. The parallel channels can have a commoninput reservoir or individual reservoirs where a collection ofmolecules, including native DNA, chromatin, RNA, proteins,polysaccharides, or small molecule drugs, is loaded. The moleculesloaded and/or analyzed can also include lipids or complexes of any ofnative DNA, chromatin, RNA, proteins, polysaccharides, or small moleculedrug. The molecules may be isolated from cells or tissues. The moleculescan be driven through the array of parallel channels by various methods,electrophoretic, electroosmotic, or pressure, and interrogated at agiven spatial location along each channel by fluorescent, electrical, orother means. Molecules can be driven at rates of several hundred orthousand per second in each channel independently. The molecules can behybridized with probes or bound with antibodies or aptamers that signifythe presence of a particular location along the molecule (e.g., a genefor DNA) or the presence of an epigenetic mark such as DNA methylation,histone methylation, histone acetylation, histone phosphorylation, etc.The interrogation of all channels can be recorded simultaneously onmeasurement devices such as photodetectors or ammeters. Based on thequantity measured in real-time during the passage of a molecule throughthe channel, each molecule can be directed toward one of severalpossible output channels. This provides for independent sorting in eachof the parallel channels, allowing molecules to be directed to one oftwo or more output channels for subsequent analysis. Such switching ofoutput channels can be accomplished by rapidly switching voltagepotentials, applying a dielectrophoretic force transverse to the flowdirection, pumping, or other means.

The channels, including downstream flow paths and channels, may have thesame of different dimensions. Downstream flow paths or channels may havethe same, higher, or lower cross-section area as compared to an upstreamchannel. The dimensions of the channels can be selected to maintainfluid velocity within the channels. In one example, an upstream channelis fluidically connected at a bifurcation point to two downstreamchannels and the cross sectional area of each of the downstream channelsis half the cross-sectional area of the upstream channel.

The devices described herein, including devices for multiplexedanalysis, can be fabricated in silicon, silicon dioxide, glass,borosilicate, polymeric, or fused silica chips using a variety ofstandard micro- and nanofabrication techniques includingphotolithography, electron beam lithography, reactive ion etching, andembossing.

The devices of the present invention may consist of channels withsubmicrometer width and height fabricated in fused silica substrates bymeans of optical lithography and plasma etching (FIG. 1). In a typicalexperiment, a fluorescent molecule is driven by electrokinetic flow(like gel electrophoresis only in the fluid phase), through the channeland excited by a focused laser beam as shown in FIG. 2. The lightemitted from the fluorophore is collected and quantified by aphotodetector. These experiments demonstrate our ability to perform highthroughput multicolor imaging and quantitative analysis of biologicalmolecules on our devices, methods readily adapted to epigenomicanalyses. For example, FIG. 3 depicts single DNA molecules labeled witha fluorescent dye flowing though the channels. This is a novel designbecause it drastically reduces the sample studied to a limit beyond thatpossible with conventional optics alone and incorporates a fluidicstructure to allow all molecules to be analyzed rapidly and equally onan individual molecule basis.

Other examples of channels and systems for analyzing and/or sorting anobject can be found in U.S. Patent Application Nos. 2009/0050542, and2009/0234202, U.S. Pat. Nos. 6,927,065, 7,405,434 and 6,833,242, and PCTPublication No. WO/2010/044932 which are each incorporated herein byreference in their entirety.

Detection Module and Light Sources

The system can comprise one or more detection modules configured tomeasure a signal corresponding to a property of the object or geneticmaterial to be analyzed. The detection modules described herein canutilize a variety of detection techniques. The detection modules candetect optical, electrical, radioactive, physical (e.g., size, density,thermal conductivity, elasticity, viscosity, and strength), and/ormagnetic properties. The choice of a detector can depend on the type oflabel used. For example, an optical detector can be used to detect afluorescent label, and a conductance meter or electrical detector can beused to detect a metallic label. Examples of electrical detectionsystems are described in PCT Publication No. WO/2010/044932, which ishereby incorporated by reference in its entirety. An electrical detectorcan comprise a wire, a nanowire, a nanotube, a transistor, or acapacitor placed in proximity to a detection zone. The electricaldetector can be made of carbon, silicon, carbon/silicon, or othersemiconducting material.

A detection module utilizing optical detection of a property of theobject can comprise a light detector or photodetector. The lightdetector can be a CCD, CMOS array, photomultiplier tube, avalanchephotodiode (APD), single photon counting modules, photoresistors,photovoltaic cells, phototransistors, LEDs, and any combinationsthereof.

The detection module can include one, two, three, four or more lightsources configured to illuminate a channel. The light sources can beconfigured to create one or more interrogation volumes. The lightsources can include lasers, LEDs, fluorescent lamps, incandescent lamps,halogen lamps, gas-discharge lamps, and/or high-intensity dischargelamps. The one or more light sources can emit one or more beams of lightthat illuminate one or more regions or volumes within the one or morechannels. The beam of light may be focused by an optical component,e.g., a high numerical aperture objective. The beams of lightilluminating the channels can create one or more inspection volumes thatare defined by the walls of a channel and the beams of light. Thedimensions of the beam of light and the channel can define the size ofthe inspection volume, as shown in FIG. 3. The inspection volumes canhave a volume that is about, up to about, or greater than about 0.01,0.05, 0.1, 0.2, 0.5, 0.75, 1, 5, 10, 25, 50, 75, or 100 femtoliters. Theterm inspection volume can also be referred to as an interrogationvolume, optical volume, or a detection volume. The interrogation volumecan be sized to hold a single object, single chromatin, or singlenucleic acid. In some embodiments, the interrogation volume can be sizedto hold only a portion of a single object, single chromatin, or singlenucleic acid.

The light source can create a beam of light that is up to about, about,or greater than about 0.1, 1, 5, 10, 50, 100, 200, 280, 300, 500, 750,1000, or 2000 μW within the interrogation volume.

In some embodiments, the plurality of light beams emit distinctspectrums or wavelengths of light into the same or different locationsin the channel. The beams of light may create overlapping interrogationvolumes or distinct interrogation volumes. The beams of light may have adiameter of about, less than about, or greater than about the width ofthe channel. In some embodiments, the beam of light is about, up toabout, or greater than about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 timeswider than the width of the channel. The width of the beam of light canbe selected such that the light within the channel is substantiallyuniform.

The detection module can also include one or more optical components.For example, the detection module can include one or more high numericalaperture objectives or lenses, optical fibers, mirrors, dichroicmirrors, gratings, filters, and confocal apertures. The arrangement ofthe light source, detectors, and optical components can allow fordetection of one, two, three, four, five, six, or more optical signals.The detection can be simultaneous or time-correlated. The resolutionand/or accuracy of the time-correlation and/or time-dependent signal canbe up to about or about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1, 5, 10, 50, 100, 500, or 1000 milliseconds. The resolution and/oraccuracy of the time-correlation and/or time-dependent signal can be upto about or about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,5, 10, 50, 100, 500, or 1000 microseconds. The resolution and/oraccuracy of the time-correlation and/or time-dependent signal can be upto about or about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1,5, 10, 50, 100, 500, or 1000 nanoseconds.

Several thousand molecules can be interrogated each second in each ofseveral thousand parallel channels. By recording the response of eachmolecule, which may be simultaneous and/or sequential, to an externalstimulus the devices described herein can analyze several orders ofmagnitude more molecules than present technology allows. The analysis isan improvement over prior technology in terms of molecules analyzed pertime, per device footprint, per reagent volume used, or any combinationthereof. The device can be used to rapidly analyze and sort a variety ofmolecules and/or complexes. The molecules and/or complexes can includeindividual DNA, RNA, chromatin, protein, polysaccharide, or small drugmolecules, or lipids, or complexes of individual DNA, RNA, chromatin,protein, polysaccharide, or small drug molecules, or lipids. Themolecules and/or complexes can be analyzed for a range of biologicallyrelevant information including binding efficiency to various probes, orthe presence of various other biological markers signifying the presenceof genes, epigenetic marks, or haplotype information.

The one or more detectors can be configured to transmit informationregarding the one or more detected signals to a processor. The processorcan be a programmable logic device, a computer, or any other componentthat can record or interpret the signal. The processor may be acomponent of a sorting module, described herein.

Processor and Computer System

The system can comprise a processor that interprets one or more signalsmeasured by the detection module and/or directs an object to one of aplurality of downstream channels or flow paths. The informationtransmitted by the detection module or the detector can be received by aprocessor. The processor can be a computer or a programmable logicdevice, e.g., a field-programmable gate array (FPGA), programmable arraylogic (PAL), generic array logic (GAL), or complex programmable logicdevice (CPLD). The processor can operate at about, up to about, orgreater than about 15, 25, 50, 60, 75, 100, 200, 500, 1000, or 2000 Hz,MHz, or GHz. In some embodiments, the processor is an FPGA that caninterpret data and return instructions in less than about 1×10⁻², 2×10⁻²1×10⁻³, 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶, or 1×10⁻⁷ seconds. The processor can beprogrammed to receive a signal and/or direct an object to one of aplurality of downstream flow paths based on the signal. The conditionsfor sorting an object to one path or another can be programmed into theprocessor by a user via a user interface on a computer system or anotherdevice. The conditions for characterizing and/or sorting an object caninclude simultaneous or time-correlated detection of two, three, four,five, six, or more signals. Detection of more one, two, three, four,five, six, or more signals above a preselected threshold can be used todiscriminate objects. In some embodiments, the signals correspond tooptical, electrical, magnetic, radioactive, or physical properties ofthe object. The signals measured can be indicative of distinctproperties of the object.

The processor can interpret the data to determine how to sort the objectand transmit instructions to a sorting actuator or transmit informationregarding properties of the object to a computer or a recording device.The processor can interpret data obtained at one interrogation volumepositioned in an upstream channel and provide instructions to a sortingactuator to direct the object to one of a plurality of downstream flowpaths at one, two, three, four, five, or more branch points. Theprocessor can interpret data obtained at one interrogation volume andreturn instructions to a sorting actuator to direct the object to one ofa plurality of downstream flow paths at a branch point immediatelyadjacent and/or downstream to the interrogation volume. The branch pointcan be a known distance, volume or time away from the interrogationvolume. Precise knowledge of the distance, volume, or time between theinterrogation volume and the branch point can allow for accurate and/orprecise sorting of the object. Reduction of the distance, volume, ortime between the interrogation volume and the branch point can increasethe precision and/or accuracy of sorting.

A sorting actuator can perform sorting of the object by changing thetrajectory or flow path of the object. The sorting actuator can alsochange the trajectory or flow path of the object or of a fluid carryingthe object. The sorting actuator can sort the object by physicallyaltering the flow paths within the channels and downstream flow paths,or by imparting magnetic, electrical, or optical forces. In someembodiments, the sorting actuator can comprise one or more electrodesthat impart an electrokinetic force on the object. Alternatively, thesorting actuator can comprise one or more valves that change the flowpath of a fluid carrying the object. The valves can be pneumatic valves,mechanical vales, magnetic valves, rotary valves, hydraulic valves,piezoelectric valves, shuttle valves, elastomeric valves, and electricalvalves. In some embodiments, electrodes can also be used to change theflow path of a fluid carrying the object. Electrostatics can also beused to alter the flow path of an object. Optical tweezers may also beused to alter the flow path of an object by placing the object in aposition to flow down one of a plurality of downstream flow paths ordeflecting the object toward one of a plurality of downstream flowpaths. Optical tweezers can be used in the invention to trap and moveobjects, e.g. molecules or cells, with focused beams of light such aslasers.

The system can be operably linked to a computer that has a userinterface for controlling sorting system (e.g., the detection module andthe sorting module), programming the programmable logic device, anddisplaying results. The computer can be an integral part or a separatepart of the subject sorting system. The user interface can be agraphical user interface. The user interface can display themeasurements by the detector, the analysis of the object, and thesorting of the object, which may be in real-time. The user interface canallow for selection of operating conditions, e.g., voltage gradient,sorting conditions, signal thresholds, total sorting time, light sourcepower, and detector calibration. The operating conditions can beinputted using a keyboard, mouse, or other input device.

In some embodiments of the invention, the sorting system comprises amemory module for storing data transmitted by the processor and/ordetector during sorting. The memory module can be accessed duringsorting or subsequent to sorting to retrieve data collected duringsorting.

The analytical system described herein can also comprise a computersystem for user interaction and data retrieval. The computer system canhave a user interface for inputting experimental parameters anddisplaying the results of the analysis. The user interface can displaydata in real-time. The computer system can have a display device fordisplaying the user interface and displaying the real-time data.

Exemplary Applications

The subject system provides an effective tool for sorting and/oranalyzing an object at a single-object level. The subject system finds avast array of applications including, but not limited to, geneticanalysis such as epigenetic analysis of chromatin, sorting of cells,nucleic acid molecules, proteins, carbohydrates, lipids, and anycombination thereof.

Detection of Quantum Dots

Fluidic channels with submicrometer dimensions of the present inventionmay be used to isolate, detect and identify individual quantum dots(QDs) conjugated with organic fluorophores. The channels may befabricated in fused silica with a 100, 200, 300, 400, 500, 600, 750 or1000 nm square cross section. The resulting focal volume ofapproximately 100-1000 attoliters reduces fluorescent background andincreases the signal to noise ratio of single molecule detection. Thechannels allow for the rapid detection of 50%, 60%, 75%, 90%, 95%, 99%,99.5%, 99.9% or more of the QDs and organic fluorophores traversing thefocal volume.

QD conjugates may be driven through the channels electrokineticallyexcited with a single wavelength laser and detected with a confocalmicroscope. Fluorescence emission may be collected simultaneously fromgreen and red regions of the spectrum. Signal rejection may be minimizedby the narrow and symmetric emission spectra of the quantum dots. Forefficient multicolor detection and characterization of single moleculebinding, QDs may be bound to Alexa Fluor 488 (AF488) molecules andindividually detected as shown in FIG. 6. The union of fluidic channelswith submicrometer dimensions and QDs as fluorescent labels results inefficient and rapid multiplexed single molecule detection and analysis.

Analysis of Methylation States

Analysis of DNA methylation states using purified mammalian DNA may beperformed using the devices and methods of the present invention.Further, the devices and methods of the present invention provide foranalysis of mammalian chromatin. Still further, the devices and methodsof the present invention allow for simultaneous analysis of multipleepigenetic marks (multiplexed analysis) to assess their frequency andcoincidence. For example, embryonic stem (ES) cell and mouse embryonicfibroblast (MEF) cell chromatin may be analyzed simultaneously forseveral marks including: DNA methylation, DNA hydroxymethylation,H3K27me3; H3K27me3 and H3K4me3; and H3K27me3, H3K4me3, bound RNApolymerase II (RNA pol II), bound SWI/SNF, and any other DNA orchromatin bound protein, or any other known epigenetic marker. Thesemarks have been implicated in each other's mutual regulation and also asfundamental, collaborating regulators of mammalian development. Anadditional component of this invention is the ability to perform themultiplexed analysis from these high abundance sources of chromatin tolow abundance sources, namely microdissected tumors, pre-implantationembryos, and other low abundance sources.

Analysis Using QD Label to Methylated DNA

The methods described above may further be used to analyze methylatedDNA of eukaryotic or prokaryotic origin using QD-anti-methyl-C antibodyand methylated DNA in a device of the present invention. QD-antibodyconjugates are passed through fluidic devices with submicrometercross-sectional dimension at a dilute concentration of approximately 10pM (or 1 ng/ul of IgG mass antibodies). A bias of +25 Volts is appliedto create electrokinetic flow. The number of conjugates observedaverages approximately 100/min Higher molecule-counting rates arepossible with higher voltages and antibody concentrations (e.g. 100,200, 300, 400, 500, 600, 700, 1000, 10000 or more). The fluorescentbursts observed are indicative of both individual QD-anti-methyl-Cconjugates (short peaks) and small bound clusters of the conjugates(taller peaks whose heights are approximately multiples of short peaks,FIG. 10).

Next, methylated DNA is labeled with YOYO-1 or TOTO-3, and a methylatedDNA detection reagent such as a QD-labeled-anti-methyl-C antibody or alabeled MBD1 and flowed the mixture through a device of the presentinvention. In this way, a two-color time coincidence experiment isconstructed to directly observe the presence of methylated sites (red,QD label) on the DNA (green, YOYO-1 label). The DNA antibody mixture isat a dilute concentration of approximately 90 pM (or 1 ng/ul of DNA).Coincidence between YOYO-1 or TOTO-3-labeled DNA and QD-anti-methyl-Cmay clearly be identified (FIG. 11).

In some embodiments of the present invention input DNAs may be isolatedfrom wild type ES cells and ES cells homozygous for a deletion of genes,e.g., Dnmt1. The Dnmt1 mutant DNAs have their total methylationdiminished to levels approximately 15% that seen in wild type cells. Lowfrequencies of coincidence between QD-anti-methyl-C and YOYO-1 signalsmay be provided when imaging DNA from the Dnmt1−/−cells. In contrast,DNA from wild type ES cells, may provide robust signals for DNAmethylation, consistent with what has been shown by standard restrictionenzyme analysis for DNA methylation in wild type ES cells. Dnmt1 mutantES cell DNA that has been treated with the SssI methyltransferase mayfurther increase the levels of DNA methylation signals.

Gene Expression Analysis

In still other embodiments, the methods and devices provided herein mayprovide for gene expression analysis of cells or tissues. The presentinvention may be suitable for gene expression analysis of samples fromany source comprising RNA or mRNA. In some cases, the present inventionmay be particularly useful for gene expression analysis of samples fromlow abundance or limited sources such as microdissected tumors,pre-implantation embryos, blastocysts, stem cells, or embryonic stemcells. In some cases, gene expression analysis may be provided bycontacting a sample comprising labeled RNA (e.g. labeled mRNA) with adevice of the present invention, flowing or propelling the RNA through asubmicrometer channel comprising a detection area and detecting thelabeled RNA. In some cases, the detection may provide single moleculeresolution. In some cases, the RNA may further be contacted with asecond label or probe or population thereof such as an oligonucleotideprobe or population of oligonucleotide probes that specifically bind toor identifies specific sequences of RNA. In some cases, the probe maycomprise a second label. In some cases, the time coincident detection ofa labeled RNA and a labeled probe may provide for gene expressionanalysis. In some cases, this time coincident detection may provide oneor more of high throughput gene expression analysis, quantitative geneexpression analysis, or gene expression analysis at single moleculeresolution.

Genome Wide Analysis

In addition to validating that methylation of mammalian DNA can bedetected with high sensitivity and specificity, it is worth noting thatthe present invention may also reveal the frequency and density of DNAmethylation that exists across a genome, such as an ES cell genome, forexample a mouse or human ES cell genome. Current estimates suggest that60-80% of the CpGs in the mammalian genome are methylated. But becauseonly 0.1% of the genome has been tested directly for DNA methylationstatus, the proportion of DNA fragments carrying methylation and thedensity of methylation per fragment is unknown. The present inventionmay reveal that information because a very large number of labeled DNAfragments may be detected and methylation density may be detected oneach fragment, which is directly proportional to the burst intensity perfragment. The devices retain their function for weeks at a time. In lesstime than it takes to run an agarose gel, the present invention providesfor reliably sampling and imaging individual DNA fragments representingthe entire mammalian genome.

DNA Sizing and Labels to DNA

Single molecule DNA fragment sizing may be performed on the devices ofthe present invention. For example, a lambda DNA marker may be digestedwith HindIII to form fragments of known lengths between approximately600 bp and 27,000 bp. The fragments may be labeled with an intercalatingdye, e.g. YOYO-1, TOTO-3, Syber Green, ethidium bromide or any othersuitable dye, and electrokinetically driven through the channel wherethey are individually interrogated by a focused laser. The molecules maybe uniformly driven at velocities up to 1 mm/s, 2 mm/s, 5 mm/s, 10 mm/s,20 mm/s, 40 mm/s, 50 mm/s, 75 mm/s, 100 mm/s, 250 mm/s, 500 mm/s orhigher allowing for analysis rates of 1,000; 2,000; 3,000; 4,000; 5,000;6,000; 10,000; 15,000; 20,000; 30,000; 50,000; 75,000; 100,000 or moremolecules per minute. Since the molecules are uniformly stained with thefluorescent intercalating dye, the number of photons emitted by eachmolecule as it flowed through the laser spot is proportional to itslength. FIGS. 5A and 5B show the number of photons (burst size)collected from the various fragment sizes as well as the measured linearrelationship between the burst size and the known fragment size.Comparison of fragment analysis using this technique is also comparedagainst gel electrophoresis, resulting in excellent agreement.

Sorting of Methylated and Unmethylated DNA

The devices of the present invention may be used to sort mixtures ofDNAs (e.g., HindIII digested lambda DNA) that are either unmethylated ormethylated by SssI, which methylates all CpGs. DNAs may be labeled witha DNA specific dye such as YOYO-1 or TOTO-3 and with a methylated DNAspecific label such as QD-anti-methyl-C. DNA fragments may then besorted according to their methylation state as reported by theQD-anti-methyl-C. At a sort rate of 4,000 fragments per minutes, nearly600 copies of each of the seven lambda HindIII fragments may be sortedin one minute. Bisulfite sequencing, may then be performed using theinput mixture, the sorted methylated DNA and the sorted unmethylatedDNA, and sequencing primers for each of the seven fragments.Pyrosequencing may also be performed on the input mixture, the sortedmethylated DNA and the sorted unmethylated DNA.

Mouse or human DNA may also be sorted by using approximately 20,000 bpfragment sizes as input. Input DNAs may be DNAs from ES cells that carrya Dnmt1 mutation and DNAs from wild type or other mutant ES cells. TheDnmt1 mutant DNAs have their total methylation diminished to levelsapproximately 15% of those seen in wild type cells, however, at thehighly repeated IAP element (>1,000 copies in the mouse genome),methylation is reduced to essentially undetectable levels. Sortedsamples may have plenty material to use for bisulfite analysis bypyrosequencing.

Mouse or human chromatin may also be sorted by the methods of thepresent invention. For example, antibody recognizing H4K20me3 and H3,each coupled to a spectrally-distinct QD, and may be used to sort thechromatin into two populations, one enriched for both marks and theother lacking the H4K20me3 mark. Real time PCR using primers specificfor the genomic region of interest may further be used to measuring thecontent of that region of interest in the two sorted populationsrelative to the unsorted input material.

ES chromatin may also be sorted using antibodies for two separateepigenetic marks, such as for example H3K4me3 and H3K27me3. These aremarks that define the bivalent state of chromatin in ES cells asdetermined by ChIP-Chip and by ChIP-Seq. After a sort that ensures atleast 1× genome coverage, the sorted material can be sequenced by anymethods known in the art including the use of a Solexa or 454 sequencingtechnique. The devices and methods herein can therefore be used toanalyze materials from low abundance sources and for multiple epigeneticmarks simultaneously.

Resolving Dendrimer-Like DNA

Two-color single molecule spectroscopy may be provided by the methodsand devices of the present invention further demonstrated for resolving“dendrimer-like DNA” labeled with different combinations of fluorophorequantity and color. These dendrimers are synthetic branched DNAmolecules. These data demonstrate the ability to perform quantitativesimultaneous multicolor analyses of DNA on devices of the presentinvention. Coincident lasers of two different wavelengths such as forexample 488 nm and 568 nm wavelengths may be focused on a submicrometerfluidic channel to perform measurements of coincident, fluorophoreemission intensity, see FIGS. 7A and 7B. The nucleic acid engineered(NAE) labels are composed of Alexa Fluor 488 and BODIPY 630/650 labeledto each Y-shaped DNA sub-unit (FIGS. 7A and 7B), which are thenassembled into larger dendrimer-like networks. Fluidic channels areconstructed from fused silica substrates for low autofluorescencebackground. The inspection volume is uniformly illuminated and analyteflow is controlled with electrokinetic drive, allowing each molecule tobe excited equally. More on resolution of dendrimer-like DNA isdescribed in Stavis et al. Detection and identification of nucleic acidengineered fluorescent labels in submicrometer fluidic channels. 2005Nanotechnology 16 S314-S323, which is hereby incorporated by referencein its entirety.

Analysis of PCR Products

Time and color coincident spectroscopy at the single molecule level mayhave practical application in the study of PCR products. Theseexperiments demonstrate our ability to perform quantitative multicoloranalyses, simultaneously, on our devices using DNAs. In this experiment,the purification of the amplicon product normally performed following aPCR reaction is eliminated. Primer molecules can be each labeled withany dyes known in the art, e.g., with either a green (Alexa Fluor 488)or red (Alexa Fluor 594) fluorophore, while amplicon productsincorporated both colors, as shown in FIG. 8A. In FIG. 8A, labeledprimers are indicated with either a light or dark shaded circle attachedto the primer. Isolation and measurement of single molecules permitprimers and amplicons to be distinguished in-situ, on the basis of thetime and color coincidence. This detection demonstrates the ultimate insensitivity, single fluorophores attached to single molecules, and atcounting rates ˜1,000 molecules/min. The ability to detect fluorescenceat this level of sensitivity indicates that epigenetic marks can bedetected on chromatin fragments with a low density of those marks.Coincidence is studied on samples from every fourth cycle of the PCRreaction, between cycles 0 and 40. The frequency of coincident events iscompared against the primer population at each measured cycle and usedto recreate the classic PCR amplification curve (FIG. 9). This methodcan also be applied to nucleic acid samples that have not beenamplified.

EXAMPLES Example 1 A Nanofluidic Platform for Single Molecule Analysis

One strategy for high throughput analysis of single molecules ofmethylated DNA and chromatin can entail time-resolved detection andspectral identification of fluorescent labels bound to individualmolecules containing epigenetic marks of interest. We flowed anddetected DNA and chromatin in a solution confined within a nanofluidicchannel. These channels reduce the optical excitation volume forfluorescent analysis, thus enabling us to interrogate individualmolecules in solutions of relatively high concentration. The nanofluidicchannels were fabricated in a fused silica substrate usingphotolithography and reactive ion etching. FIG. 1C a shows a singlenanofluidic channel made by this process with cross-sectional dimensionsof approximately 250 nm wide by 500 nm deep. We formed 27 separatefluidic channel arrays, each with 16 parallel channels, on a 100 mmdiameter wafer. Each array also had access ports with a reservoir at theends of the channels, which we used to load the samples and to insertelectrodes for controlling electrokinetic flow.

To analyze individual molecules of DNA and chromatin flowing throughchannels, we mounted the silica wafer on a laser confocal microscope andilluminated the samples flowing through an individual channel with twooverlapping Gaussian shaped laser profiles, each with a diameter ofapproximately 1.3 μm (FIG. 17). The laser profiles were larger than thechannel width, so that every fluorescent molecule was interrogated withthe same illumination profile. The illuminated inspection volume withinthe channel was 0.16 fL. A burst of fluorescence, associated with thedifferent labeled components, was emitted as each molecule passedthrough the illumination profile. The colors composing a molecule'ssignature were separated with optical filters and then detected withavalanche photodiode detectors. A record of the red and greenfluorescence bursts were accumulated at 100 kHz onto a personal computerand then analyzed with a custom Matlab algorithm to identify singlemolecule detection (SMD) events.

Example 2 Nucleosome Detection on Native Chromatin

This experiment demonstrates that chromatin can be directed through ananofluidic channel and remain intact, having nucleosomes remainattached on the DNA. For this test we used chromatin extracted from HeLacells bearing a transgene expressing an H2B-GFP (green fluorescentprotein) fusion protein. H2B-GFP incorporated into nucleosomes allowedthe chromatin to fluoresce. We prepared native chromatin from the cellsusing standard methods, treating isolated nuclei with micrococcalnuclease (MNase) and then extracting the soluble native chromatin usinga high salt buffer. We next labeled the DNA within our chromatinpreparations with TOTO-3, a red nucleic acid stain that is spectrallydistinct from GFP. We wanted to analyze this dual labeled chromatin fortwo reasons. First, it permitted us to determine whether chromatinremained intact during nanofluidic electrokinetic flow, which isessential for any successful application of this method for epigeneomicanalysis. Intact chromatin produce time-coincident TOTO-3 and GFPfluorescent SMD events that indicate DNA and histones are bound. Second,demonstrated that simultaneous, multi-color detection of chromatin wereperformed at high rates.

FIG. 18 illustrates a 0.25 s SCAN using chromatin extracted after a 5min MNase digestion and then driven through a nanofluidic channel at 50V. The top panel shows photon bursts corresponding to TOTO-3, whichmarks the DNA, while the bottom panel shows GFP fluorescence that marksthe H2B. In order to identify the single molecule peaks shown (FIG. 18),successive photon arrival times differing by less than 200 μs weregrouped as a burst. This time is about one-third that required for amolecule to pass through the inspection volume. Bursts with a total of10 or more photons were designated as SMD events. The mean noisedetected with buffer only was 0.22 photon/50 μs. A velocity of 2.2 mm/swas calculated by fitting a histogram of the time duration (FIG. 21) forTOTO-3 SMD events. Since the sample solution was loaded into thereservoir connected to the negative electrode, only molecules carrying anet negative charge could be driven into the channel. In the pH 8.0buffer used, only histone-free DNA and intact chromatin carry netnegative charge. The net positive charge of individual free histones,each greater than 16.5 at this pH (see Supporting Information), causedthem to remain in the loading reservoir. The time-coincident SMD eventsshown in FIG. 18 are marked by shaded vertical bars spanning the plots,the majority of which are intact chromatin. Accurate identification ofdual labeled chromatin was ensured by using optical filters thatachieved more than 20 dB of spectral isolation of green GFP bursts fromred TOTO-3 bursts, essentially eliminating fluorescence spectrumcross-talk or bleed-through during detection.

Approximately, ninety-three percent of all GFP molecules identified werecoincident with a DNA molecule, indicating chromatin remained intactduring electrokinetic flow within our nanofluidic device. This degree ofcoincidence was achieved using TOTO-3 at a 1:5 dye to base pair ratio,which provided the highest fluorescence intensity while not dissociatinghistones from the chromatin. Additional measurements (Table 2 in Example15) performed with 1:10 and 1:15 labeling ratios, produced similarlevels of coincidence, further demonstrating that this labeling did notdissociate histones. We observed that approximately one-third of theTOTO-3 signals were not coincident with GFP. This population ofmolecules is expected to arise from undigested and histone-free linkerDNA known to exist in the genome, and also from intact chromatin thatcontained only the endogenous unlabeled H2B; half of the H2B in thecells is made from the GFP fusion transgene.

To further test the authenticity of chromatin detection using SCAN, weprepared chromatin from mixtures of nuclei from wild-type HeLa cells andHeLa cells with the H2B-GFP transgene, maintaining a constant total cellquantity for all mixtures. We anticipated that the rate of coincidenceshould drop with increasing amounts of wild-type chromatin, yieldingfewer fragments with H2B-GFP. Fragments from each mixture were thendetected by SCAN to determine the fraction of two-color labeledchromatin present in each mixture. We compiled (FIG. 19 a) a record ofall coincident SMD events observed during a period of 15 min using atime coincidence histogram (TCH). The TCH contains the time-offsetbetween all identified GFP events and TOTO-3 events within a fixed timewindow. The area under the peak and above the background level,describes the total chromatin fragments with H2B-GFP, which increasedwith the proportion of GFP-HeLa nuclei. We found the proportion ofchromatin fragments with H2B-GFP to exhibit a linear increase withGFP-HeLa proportion for both the 5 and 15 min digestion series (FIG. 19b). The direct proportion between chromatin fragments with H2B-GFP andinput of GFP-HeLa nuclei validates the authentic detection of chromatin.Note that the slopes differ for the two digestion times with theprolonged digestion producing a smaller slope. The coincidence per DNAwas principally reduced due to an increase in mononucleosome fragmentsand linker DNA fragments produced at longer digestion times, for aconstant number of H2B-GFP nuclei within a mixture. This trend was alsoconsistent with other chromatin preparations, wherein longer fragmentsdemonstrated a higher coincidence proportion.

We observed variation in the size of chromatin fragments prepared duringdifferent batches of MNase digestion. For example, GFP-HeLa chromatinextracted after a 5 min MNase digestion, but prepared from differentbatches of GFP-HeLa cells on separate dates yielded different fragmentsizes, as verified by gel electrophoresis (FIGS. 7 and 8). We attributethis to variation in MNase activity. As a result, nanofluidic SCANdetected a 93% coincidence per GFP for samples with a median fragmentsize above 2 kbp (5 min lane in FIG. 23); while 50% coincidence per GFPwas observed for samples with a median fragment size less than 2 kbp(100% GFP, 5 min lane in FIG. 24). Further SCAN with chromatin digestedto less than 1 kbp fragments (100% GFP, 15 min lane in FIG. 24)demonstrated 35% coincidence per GFP. The variation, if it occurs forshortened may be mitigated using a different fluorescent labelingmethod, extending SCAN to the study of short nucleosome fragments.

To evaluate the throughput of our nanofluidic device (Table 1), weutilized SCAN measurements of the 5 and 15 min digestions of 100%GFP-HeLa nuclei. The rates for SMD of both DNA and H2B were averagedover 15 min of analysis. We calculated the detection rate fortime-coincident molecules of dual-labeled chromatin by performing abackground-corrected TCH analysis for each minute of SCAN. Thethroughput for all molecule types was consistently higher for chromatinfrom the 5 min digest, as compared to the 15 min digest, and suggested ahigher sample concentration. We verified this observation in-situ byfitting a histogram of the time between each SMD event of a given colorwith an exponential model to calculate the sample concentration (FIG.22). The average fragment size was estimated separately using gelelectrophoresis (FIGS. 7 and 8) and then combined with the DNA detectionrate to derive the analysis throughput.

TABLE 1 Nanofluidic SCAN Throughput for 100% GFP-HeLa Samples MNaseTreatment (min) 5 15 DNA Concentration (pM)^(b) 588 ± 8  248 ± 2 Dual-Labeled Chromatin (molecules/min)^(a) 1067 ± 114 201 ± 36 H2B(molecules/min)^(a) 2116 ± 143 568 ± 67 DNA (molecules/min)^(a) 6238 ±611 2287 ± 184 Gel Estimated Average Fragment Size (bp)^(c) 1600 500 DNAThroughput (Mbp/min)^(d)  10  1 ^(a)Analysis for a 15 minute SCAN at anelectrokinetic drive of 50 Volts. ^(b)DNA concentration measured in-situbased upon the time between SMD events ^(c)Fragment size estimated bygel electrophoresis ^(d)DNA detection rate and estimated fragment sizeare used to calculate DNA throughput.

Example 3 Detection of DNA Methylation

Probes that have been used in epigenomic analysis were used to detect anepigenetic mark on our nanofluidic platform. We analyzed DNA methylationand used MBD1 as our probe, which has been shown to bind methylated DNAspecifically. Our test material was HindIII digested lambda DNA from amethylation deficient host, which we left unmethylated, or methylated invitro using SssI DNA methyltransferase. SssI can methylate all 3,113CpGs in the 48.5 kbp genome. We verified the effectiveness of themethylation reactions by digesting the DNA with the methylationsensitive restriction enzyme HpaI (FIG. 25). Both DNA samples werestained with TOTO-3 and incubated with MBD1, which we labeled with AlexaFluor 488. Alexa Fluor 488 is spectrally similar to GFP. The Alexa Fluor488 labeled MBD1 retained its specificity for methylated DNA (FIG. 26).To facilitate optimum binding to methylated DNA, we added a molar excessof MBD1 to the stained DNA. We found that dilution of this mixture intoour 1×TBE-based buffer resulted in stable electrokinetic flow, asindicated by consistent SMD rates and low-levels of non-specificinteraction between probes and the nanofluidic structure.

FIG. 20 a illustrates the number of coincident SMD events for MBD1 mixedwith unmethylated DNA (top panel) or methylated DNA (bottom panel). Eachmixture was analyzed for 15 min at applied potential of 100 V. Thebackground level of coincidence events in the unmethylated DNA samplewas due to an excess of probe present in the mixture. However, thecentral Gaussian peak in the methylated DNA sample showed that boundMBD-DNA complexes were detected above the high background. Wheredesired, fine tuning the subject methods can be done by incorporatingother methods known in the art to reduce background signal from freefluorescent probes.

Similar to the chromatin analysis, we verified the authenticity of thesedetection events. We prepared a dilution series of methylated andunmethylated DNA. We expected that with diminishing amounts ofmethylated DNA, we should observe a diminishing frequency of coincidentevents. This is consistent with our observed results (FIG. 20 b). Therewas a linear increase in the number of MBD-DNA complexes with increasingmethylated DNA concentration, verifying the specificity of the signalsdetected by MBD1, and demonstrating the utility of nanofluidic SCAN fordetecting epigenetic marks on individual molecules.

We have described the development of SCAN using a nanofluidic platformto analyze individual molecules using the same fluorescently labeledprobes that have been used in bulk epigenetic analysis in molecularbiology. Confinement using nanofluidic channels allowed for singlemolecule analysis to be performed within the 100-1000 pM concentrationrange, which was essential for maintaining chromatin structure. Weverified through multi-color SCAN that core histone octamers remainedbound as nucleosomes within this nanofluidic environment duringelectrokinetic flow. Dilution of wild-type HeLa and GFP-HeLa nuclei wasused to confirm the authenticity of coincidence detection. We observedchromatin fragments with a throughput of about 10 Mbp per minute using asingle fluidic channel, indicating we could SCAN the entire genome of afungal model organism in as few as eight minutes. We envision scalingour current throughput using parallel and/or radial arrays with tens orhundreds of fluidic channels to perform analysis of larger organisms.For example, using 10 fluidic channels would allow 1× coverage of a 3Gbp human genome to be scanned in just 30 minutes.

Further reduction of the nanofluidic channel cross-section would allowfor SMD at higher concentrations and increase the signal to noise ratiofor single molecule fluorescent analysis. Since the probability ofdetecting a coincident event randomly is related to the sampleconcentration in the inspection volume, this probability can beengineered by reducing the channel cross-section. With sufficientlynarrow channels the flowing chromatin or DNA molecules can be elongatedallowing multiple fluorescent labels to be spatially separated andresolved. This may permit molecular mapping with spatial resolutionsufficient to identify multiple epigenetic marks on a single nucleosomeor to distinguish marks on adjacent nucleosomes. Prior work has shownoptical resolution of molecular length to 114 nm, equivalent to about335 bp, during rapid flow of lambda DNA. This spatial resolution waspossible using nanofluidic channels with cross sectional areas on theorder of 0.01 μm², an order of magnitude less than was used in theseexperiments. It is likely that smaller channels will allow us toidentify molecules with multiple bound probes and resolve theirpositions on a chromatin fragment.

Example 4 Calculation of Histone Charge

The amino acid sequence for each of the four core histones were enteredinto an online calculator. The solver provided the charge for each corehistone at various pH values and 16.5 was the smallest positive chargeassociated with a free histone at pH 8.0.

Example 5 Preparation of HeLa Cell Chromatin

HeLa cells constitutively expressing green fluorescent protein onhistone H2B (H2B-GFP) were cultured as monolayers in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 5% fetal calf serum. Nativechromatin fragments were prepared from HeLa cells as follows:

Briefly, cells from two 15 cm plates were scraped and washed once with1XRSB (10 mM Tris pH 7.6, mM NaCl, 1.5 mM MgCl₂), resuspended in 5 mL1XRSB buffer with 1% Triton-X 100 and homogenized using a Douncehomogenizer fitted with a loose pestle using eight strokes. Nuclei wererecovered by centrifugation, resuspended in 1.5 ml Buffer A (15 mM TrispH 7.6, 15 mM NaCl, 60 mM KCl, 0.34 M Sucrose, 0.5 mM Spermidine, 0.15mM Spermine, 0.25 mM PMSF and 0.1% β-mercaptoethanol), to which we added15 μL 0.1 M CaCl₂ and 1.5 μL of micrococcal nuclease (3 units/μL) andincubated for various times, using 5 μL of 0.5 M EDTA to stop thedigestion. Digests were centrifuged, the supernatants discarded, andeach 140 μL aliquot of nuclei were resuspended in 450 μL 10 mM EDTA, 50μL 5 M NaCl to solubilize the chromatin. Soluble chromatin was separatedfrom the insoluble debris by centrifugation. For gel analysis of DNA, wetook 60 μL of chromatin, added 24 μL H₂O, 6 μL 10% SDS and 24 μL 5 MNaCl, then extracted DNA with a 1:1 mixture of phenol-chloroform and 10μL supernatant was analyzed by 1.5% agarose gel electrophoresis. We useda fluorimeter to measure the fluorescence of the intact chromatin and aspectrophotometer to measure the amount of DNA extracted from thechromatin. This served to calibrate the fluorimeter reading, allowingdirect estimation of the sample concentration during subsequentchromatin isolation preparations without removing histones. Thecalibration was reproducible between preparations.

Probe selection and labeling can be fundamental to SMD. Our initialattempts to detect chromatin signatures used antibodies. Because theyhave two antigen binding sites, a single antibody molecule cancross-link two chromatin fragments. At antigen and antibodyconcentrations that allow the classical precipitin reaction, aggregatesform, which can clog the nanofluidic channel. This can be avoided byusing vast antibody excess, or monovalent probes for epigenetic marksincluding Fab fragments of antibody, aptamers, or monovalent probes suchas the MBD1 protein we used carrying a single methyl binding domain.

Example 6 Methyl Binding Domain (MBD1) Protein Synthesis and Labeling

Plasmid for bacterial expression of 1×MBD (pET-1×MBD) was provided byAdrian P. Bird at The Wellcome Trust Centre for Cell Biology, Universityof Edinburgh, UK. Recombinant His6-tagged MBD1 was purified from 100 mLinduced BL21 (DE3) cultures on Ni-NTA agarose (Qiagen) usingdenaturation and on column renaturation cycles in accordance with themanufacturer's instructions, with some modifications.

The methylation sensitive binding domain (MBD) from MBD1 was cloned intothe pET-30b plasmid in order to create a His-tag fusion protein that wasexpressed under IPTG induction. IPTG induced E. coli were lysed and thefusion protein was purified and refolded on a nickel column. Followingpurification the fusion protein was labeled with Alexa 488 usingInvitrogen's microscale labeling kit (A30006), which labels free amines.Three additional rounds of resin purification were preformed to betterpurify away free labels. The integrity of the labeled MBD probe wasassayed with a slot-southwestern blot (FIG. 26). Though absorbancemeasurements indicated an average degree of labeling of 1.5 dye per MBD,we believe that three subpopulations remained within the labeledmixture: dye-labeled MBD, free unbound dye, and MBD with no dye. Ifthree subpopulations exist within the dye-labeled MBD solution, thenumber of bound MBD-DNA complexes would be under-reported.

Example 7 Lambda DNA Preparation and In-Vitro Methylation

Lambda DNA from phage grown in a methylation deficient host (PromegaD1521) was digested with HindIII and methylated in vitro with SssImethyltransferase, which can methylate all 3,113 CpGs in the 48.5 kbpgenome. Efficacy of the methylation reaction was assessed by resistanceto digestion by the methylation sensitive enzyme HpaII.

Example 8 MBD-DNA Affinity Reaction

In a mixture of methylated and unmethylated DNA suspended at 50 ng/μL in1× Tris Buffered Saline (1×TBS, 50 mM TRIS-HCl and 138 mM NaCl and 2.7mM KCl, pH 8.0), we performed DNA staining using TOTO-3. Approximately 1μg of labeled DNA was then diluted into 20 μL of buffer containing 1×TBSwith 2% bovine serum albumin and 0.1% TritonX-100 (v/v). A volume of 1μL MBD1, stored at 280 ng/mL, and labeled with AlexaFluor488 was thenadded to the DNA to perform the binding reaction under conditions ofmolar excess. The binding reaction occurred for 2 hours at roomtemperature.

Example 9 DNA Labeling

We use the cell-impermeant, intercalator TOTO-3 (Invitrogen). Thelabeling reaction was conducted by mixing the diluted DNA or chromatinand the diluted dye according to the method described by themanufacturer. All samples were prepared with a 1:5 dye to base pairratio, unless otherwise noted. Following the labeling reaction, sampleswere protected from light and stored overnight at 4° C. TOTO-3 exhibitssignificant fluorescence enhancement upon binding, which alleviated theneed for purification to remove unbound dye following the labelingreaction.

Example 10 Fabrication of Nanofluidic Channels

Nanofluidic channels were fabricated in a fused silica substrate.Projection photolithography (GCA Autostep 200) was used to patternfluidic channels with a 500 nm critical dimension. This method allowedrapid patterning of 27 fluidic channel arrays, totaling 432 fluidicchannels, on a 100 mm diameter wafer. Patterns formed in the developedphotoresist were transferred approximately 250 nm into the silica usingreactive ion etching (Oxford 80, Oxford Instruments). The fluidicchannels were protected with photoresist during subsequent through-waferdrilling using a focused jet of alumina abrasive to form the accessports at the ends of the channels. The wafer surface was cleaned with ahot Piranha solution (3 H₂SO₄:1 H₂O₂) and RCA standard clean (5 H₂O:1NH₄OH:1 H₂O₂, heated to 70° C.).

Direct touch bonding with a 170 μm thick coverslip wafer was performedto cap the fluidic channel. A subsequent high-temperature anneal to1050° C. permanently bonded the stack of fused silica wafers together.An optical-grade epoxy (Norland Products) was used to attach fluidreservoirs to the wafer surface.

Example 11 Electrokinetic Flow in Nanofluidics

Samples were kept in their respective storage buffer until DNA labelingand/or MBD binding reactions. Following these reactions, samples wereserially diluted in 1× Tris-Borate-EDTA (1×TBE, 89 mM TRIS borate and 2mM EDTA, pH 8.0), with additives 0.5% polyvinylpyrrilidone (PVP)measured (w/v) and 0.1% TritonX-100 measured (v/v) (both from SigmaAldrich, St. Louis, Mo.). The final dilution suspended the samples at anestimated concentration of 600 μM, nominally 1-2 ng/μL for chromatinsamples, and about 0.25 ng/μL for methylated DNA samples. The polymeradditives in the buffer served to limit electroosmotic flow and preventnon-specific interactions with the fluidic channel walls withoutdenaturing proteins. We loaded 50 μL of the sample solution into theinput reservoir of a fluidic channel array and then connected to thenegative electrode. The output reservoir contained only the buffersolution and was connected to the positive electrode. Samples wereflowed at an applied bias of 50 V for all chromatin samples and 100 Vfor all methylated DNA samples. Stable electrokinetic flow wasestablished during a pre-flow time of 20 min to ensure steady-state flowconditions had been achieved prior to data collection. Each sample wasexamined for a total of 15 min, always using the same fluidic channelwithin the array. Following single molecule detection, the fluidicchannel array was rinsed iteratively for a total of 30 min and thenchecked to verify the absence of fluorescently-labeled molecules, priorto loading the next sample. Fluid channel arrays used with chromatinexperiments were not reused with DNA methylation experiments.

Example 12 Laser Induced Fluorescence Confocal Microscopy

Single molecule fluorescence was observed using an inverted microscope(IX-71, Olympus) equipped with a side laser port. Laser illumination of330 μW at 488 nm (Sapphire, Coherent) and 1300 μW at 635 nm (Cube,Coherent) were overlapped in free-space, incident on a dual-banddichroic mirror (488/647rpc, Chroma Technology) and then focused ontothe nanofluidic channel using a 60×, 1.2 numerical aperturewater-immersion objective (UPlanSAPO, Olympus), and aided with anelectron multiplied CCD camera (Cascade 512B, Photometrics). A dual-bandlaser notch filter (NF01-488/647, Semrock) attenuated stray laser lightand passed single molecule fluorescence. Confocal spatial filteringoccurred using a 50 μm diameter pin-hole (901PH, Newport). Two colorfluorescence was then chromatically split using a second dichroic mirror(FF560-Di01, Semrock) and then filtered by band pass fluorescencefilters (525/50M and 680/40M, Chroma). Each color of fluorescence wasthen collected using a 100 μm diameter core multimode optical fiber (OZOptics). Photons were detected by avalanche photodiodes (APD) in singlephoton counting mode (SPCM, Perkin-Elmer) and recorded at 100 kHz usinga high-speed correlator (correlator.com) and a personal computer.

Example 13 Statistical Analysis

Propagated error analysis was performed to evaluate the proportion ofbound molecules, chromatin or MBD-DNA, with respect to total DNA. First,we defined a window region that encompassed the full width of theGaussian distribution in a time coincidence histogram. Adjacent to thewindow region were the sidebands, which were used to characterize thebackground of uncorrelated molecules. The background contribution in thewindow region was calculated based upon the uncorrelated molecules per50 μs bin in the sidebands, reported as the mean and standard deviation,and then scaled by the number of bins within the window region. Thetotal molecules counted within the window region was summed and reportedwith a Poisson counting error. The number of bound molecules was thencalculated by subtracting the total molecules from the backgroundmolecules within the window region and propagating the error. Second,the number of total DNA molecules observed was counted and reported witha Poisson counting error. The ratio of bound molecules and total DNAmolecules was then calculated and the errors of each were propagated. Asapplicable, we plotted the average value and error bars that representedthe propagated error.

Fitting error analysis was performed to measure the concentration ofmolecules measured within a nanofluidic channel. The inter-event timeseparation was plotted as a histogram and then fitted to single-termexponential decay using Matlab's built-in fitting routine. The fittedmean inter-event time was calculated with a 95% confidence interval. Themean and confidence interval were then evaluated using a Poisson model,to describe molecule occupancy within the inspection volume, to reportconcentration values.

Example 14 Calculation of Burst Duration and Molecule Velocity

An applied voltage created an electric field within the nanofluidicchannel that transported the charged molecules through the inspectionvolume. The burst duration describes the transit time for a molecule topass through the inspection volume. Here we examine the burst durationfor chromatin molecules prepared with a 5 min MNase digestion from 100%GFP-HeLa nuclei and driven through a channel at 50 V. This data is asubset of those shown in FIG. 19 and was prepared from the chromatinpreparation shown in FIG. 24. The burst duration (t) for eachfluorescent dye color was examined separately and then fitted with aGaussian model with free parameters μ, for the mean burst duration, andσ, corresponding to standard deviation in the burst duration. Thefitted, mean burst duration is provided on each plot with a 95%confidence interval. The Gaussian model used was:

$f = \frac{{\mathbb{e}}^{- {(\frac{t - \mu}{\sigma\sqrt{2}})}^{2}}}{\sqrt{2{\pi\sigma}^{2}}}$

Given the approximate laser beam diameter (x_(laser)) of 1.3 μm and themean burst duration (t_(burst)) of 585 μs and 495 μs for the DNA and H2Bmolecules, respectively, we calculated a mean velocity of 2.2 mm/s and2.6 mm/s using

$v = {\frac{x_{laser}}{t_{burst}}.}$See also FIG. 21.

Example 15 Intercalator Effects on Nucleosome Integrity

The red DNA stain, TOTO-3, was added to GFP-HeLa chromatin in varyinglabeling ratios to examine the effects of intercalation on nucleosomeintegrity. Chromatin prepared with a 5 min MNase digestion (FIG. 23) wasdiluted to approximately 1 ng/mL and driven at 50 V through ananofluidic channel. We used SCAN to analyze the number of DNA moleculeslabeled with TOTO-3 and the number of H2B molecules labeled with GFP forthree different labeling ratios, 1:5, 1:10, and 1:15 dye to base pair.The results are shown in Table 2 below.

TABLE 2 SCAN analysis of DNA molecule labeling TOTO-3 DNA-TOTO3 H2B-GFPCoincident Coincident Coincident Labeling Molecules Molecules MoleculesMolecules:DNA Molecules:H2B None 2678 ± 52 21828 ± 148 1128 ± 122 0.421± 0.0462  .052 ± 0.00560 1:15 36349 ± 191 11569 ± 108 9604 ± 431 0.264 ±0.0526 .830 ± 0.0381 1:10 105381 ± 325  37372 ± 193 32168 ± 1232 0.305 ±0.0117 .861 ± 0.0333 1:05 41898 ± 205 15819 ± 126 14803 ± 325   0.353 ±0.00795 .936 ± 0.0219

We observed approximately 93% coincidence for the 1:5 labeling ratio,which is recommended by Invitrogen for proper intercalation. At the 1:10and 1:15 labeling ratio, we observed a slightly reduced level ofcoincidence due to the reduced intercalation efficiency. Since allsamples were examined in the same fluidic channel and the TOTO-3 labeledsamples were examined first, we observed a small level of TOTO-3adsorption to the fused silica surface of the channel. This adsorbed dyewas subsequently collected by unstained chromatin and resulted in a 5.2%coincident detection rate.

Example 16 Calculation of Burst Separation and In-Situ SampleConcentration

For the dilute sample concentrations used in these experiments, we wereable to track the burst separation. The burst separation describes thetime elapsed between bursts and is related to the concentration of thesample analyzed. Here we examined chromatin molecules prepared with a 5min MNase digestion from 100% GFP-HeLa nuclei and driven through achannel at 50 V. This data is a subset of those shown in FIG. 19 and wasprepared from the chromatin preparation shown in FIG. 24. The burstseparation (t) for each fluorescent dye color was examined separatelyand then fitted with an exponential model with free parameters β, λ. Thefitted, mean burst separation is provided on each plot with a 95%confidence interval. The exponential model used was:f=βe ^(−λt) where λ=CAv

The data is shown in FIG. 22. In this model, β is a scaling factor. Thefitted parameter λ was related to ‘A’ is the cross-sectional area of thechannel (0.125 μm²) and ‘v’ is the flow speed of the molecules tocalculate ‘C’, the concentration of molecules. The flow speed wasderived using the methods outlined with FIG. 21, which resulted in anin-situ measured concentration of 588±8 pM for the DNA and 248±2 pM forthe H2B, as given in Table 2.

Example 17 DNA Fragment Sizing by Gel Electrophoresis

Chromatin was extracted from HeLa nuclei and digested using MNase. Withincreased duration MNase treatment, we observed a decrease in chromatinfragment sizes, as verified by gel electrophoresis. Batch-to-batchvariations in preparation, attributed to MNase activity, resulted indifferent range of fragment sizes for the same digestion time. Forexample, we observed the 5 min digestion (FIG. 23) to yield fragmentlengths well-above the 2,072 bp marker. However, a series of digestionsperformed on a later date (FIG. 24) yielded a more disperse range offragments, nominally centered between 1-2 kbp.

Example 18 In-Vitro DNA Methylation Test with HpaII

Lambda DNA prepared from phage grown in a methylation deficient host(Promega D1521) was left unmethylated or methylated in vitro with SssImethyltransferase. As a test for efficacy of the methylation reaction,aliquots of DNAs were then digested with HindIII or with both HindIIIand the methylation sensitive enzyme HpaII. Resistance to digestion byHpaII is evidence for DNA methylation. Base pair sizes of HindIIIfragments are shown in FIG. 25.

Example 19 Southwestern Blot Analysis of MBD-1 Activity and MethylationSpecificity

Both unmethylated and in-vitro methylated lambda DNA were bound to anitrocellulose membrane in varying quantities using a slot blottingapparatus. The Alexa 488 labeled MBD1 protein was then used to probe theentire blot. Following over night incubation at 4° C. the blot waswashed and scanned with Typhoon imager to detect the Alexa Fluor 488label. The results are shown in FIG. 26. This image demonstrates thatthe MBD1 probe binds to methylated DNA with high specificity.

Example 20 Parallel Fluidic Channels for High Throughput MultiplexedMolecular Analysis and Sorting

The present invention provides for methods and devices having an arrayof parallel fluidic channels, where each channel has a width and depthless than one micron and a length of a few millimeters. This arrangementcan allow for the interrogation of thousands of molecules per second ineach of a plurality of channels. The number of channels that arearranged in a device can be about, greater than about, or up to about 1,40, 100, 500, 1,000, 5,000, or 10,000.

For example, FIG. 28 shows a schematic illustration of parallel fluidicchannels with multiple input reservoirs. The channels have an opticalinterrogation region where lasers or other near field excitation sourcescan excite fluorescent labels attached or bonded to single biomolecules.Alternatively, the parallel channels may have a common input reservoiror individual reservoirs where a collection of molecules, includingnative DNA, chromatin, RNA, proteins, polysaccharides, or small moleculedrugs, is loaded.

FIG. 29 shows a schematic illustration of parallel fluidic channels withmultiple output reservoirs. As indicated, each channel has two potentialoutput channels that molecules can be switched between depending on themeasured value of some fluorescent or electrical or other property ofthe molecule detected when it traversed the excitation volume. As shownin FIG. 30, each of the parallel input channels can be interrogated atmultiple locations leading to multiple possible output paths based onthe measurements made at each interrogation zone. As shown in FIG. 30,four potential output path channels may be connected to each parallelinput channel.

Alternatively, the parallel channels may also have a common outputreservoir where the analyzed molecules are deposited.

The devices for detection can be configured to analyze the plurality ofchannels in a variety of manners. For example, FIG. 31 shows a schematicillustration of a fluidic chip interrogated optically with a lens forimaging the resulting fluorescent emission from each channelindependently on a CCD, CMOS array or other arrayed photodetector. Theschematic illustrates a diffractive element that separates differentwavelength emission fluorescence transverse to the incoming excitationlight, allowing each channel to be imaged on the photodetector along aseparate axis. This allows for multiplexed analysis of each biomolecules(detection of several separate probes indicating the presence of variousbiological marks) for each parallel fluidic channel.

A method of fabricating a device having a plurality of channels is shownin FIG. 32. Referring to FIG. 32, the bottom substrate can be made offused silica that has been patterned using nanolithography ormicrolithography techniques to include thousands of parallel fluidicchannels. The bottom substrate can be bonded to a second fused silicawafer with access holes for fluidic reservoirs. The second fused layercan have a PDMS gasket on top for bonding to a final glass layercontaining larger ports and integrated electrical contacts forcontrolling electrophoretic flow within the channels.

A scanning electron micrograph of an exemplary device is shown in FIG.33. As shown in FIG. 33, each channel is approximately 150 nm wide anddeep with a length of ˜100 microns. The channels are made by reactiveion etching fused silica after it being patterned with a negative toneelectron beam resist. The wafer is subsequently bonded to a cover wafercontaining reservoir holes where DNA molecules can be introduced andelectrophoretically driven through the channels.

FIG. 34 shows a schematic of a device having common input and outputreservoirs and a central region of 40 parallel nanofluidic channels.This device does not contain separate sorting capability for eachchannel, as described elsewhere herein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for performing epigenetic analysis ofnative chromatin in a channel, comprising: (a) flowing the nativechromatin through said channel, wherein said native chromatin is labeledwith a plurality of labels, at least one of which is specificallycomplexed with an epigenetic marker on said native chromatin, and atleast one other label is complexed with a protein and/or nucleotide ofsaid native chromatin; (b) illuminating the channel using one or morelight sources to create a plurality of interrogation volumes along saidchannel, wherein each interrogation volume is defined by walls of asubmicrometer channel region that are separated by a width of less thanabout 1 μm of said channel and a beam of light; (c) detecting the atleast one label and the one other label from the same or distinctinterrogation volumes of said plurality to generate time-correlatedresolution of said at least one and said at least one other label; and(d) analyzing said time-correlated resolution, thereby performing saidepigenetic analysis.
 2. A method for displaying real-time epigeneticsdata regarding an native chromatin in a channel, comprising: (a) flowingthe native chromatin through an illuminated interrogation volume,wherein said illuminated interrogation volume is created by using a beamof light to illuminate a submicrometer channel region of said channelhaving walls that are separated by a width of less than about 1 μm anddefined by said walls and said beam of light, and wherein said nativechromatin is labeled with a plurality of labels, at least one of whichis specifically complexed with an epigenetic marker on said nativechromatin, and at least one other label is complexed with said nativechromatin; (b) detecting the at least one label and the at least oneother label within the interrogation volume in real-time, therebyproducing said real-time epigenetics data; and (c) displaying saidreal-time epigenetics data on a display device, wherein said real-timeanalysis data reflects information on said at least one label and saidat least one other label.
 3. The method of claim 1 wherein the pluralityof labels comprise at least three or four labels.
 4. The method of claim1 wherein the at least one other label is complexed with a histone or isa nucleic acid binding agent selected from the group consisting ofsequence specific probe, intercalating dye, minor groove binder, and DNAbinding proteins.
 5. The method of claim 1 wherein the at least oneother label is complexed with a binding agent that complexes with atarget selected from the group consisting of a non-histone protein, atranscription factor, MBD1, RNA Pol II, and RNA.
 6. The method of claim1 wherein the detecting step provides a time-dependent resolution ofbetter than about 10 microseconds.
 7. The method of claim 1 wherein thenative chromatin comprises a nucleic acid molecule and a histone.
 8. Themethod of claim 1 wherein the illuminated interrogation volume(s)contains a single chromatin.
 9. The method of claim 1 wherein theinterrogation volume(s) is/are less than 0.5 femtoliters.
 10. The methodof claim 1 wherein the native chromatin is characterized in less thanabout 0.1 seconds.
 11. The method of claim 1, wherein the at least onelabel and the one other label are detected simultaneously.
 12. Themethod of claim 1, wherein the at least one label and the one otherlabel are detected sequentially.
 13. The method of claim 1, furthercomprising (d) flowing the native chromatin-through a second channel,wherein the channel and the second channel are different channels; (e)illuminating the second channel using one or more second light sourcesto create a plurality of second interrogation volumes along saidchannel, wherein each second interrogation volume is defined by walls ofsaid second channel and a second beam of light; and (f) detecting the atleast one label and the one other label from the same or distinctinterrogation volumes of said plurality of second interrogation volumes.14. The method of claim 13, wherein the native chromatin is deliveredsimultaneously or sequentially to the channel and the second channel.15. The method of claim 1, further comprising directing the nativechromatin to one of two or more output channels, thereby sorting thegenetic material.
 16. The method of claim 1, wherein the at least onelabel which is specifically complexed with an epigenetic markercomprises an antibody or aptamer.
 17. The method of claim 2, wherein thesubmicrometer channel region walls are separated by a width of less than1 μm and a height less than about 1 μm.
 18. The method of claim 2,wherein the native chromatin comprises nucleic acid and a histoneprotein.
 19. The method of claim 1, wherein the submicrometer channelregion walls are separated by a width less than about 1 μm and a heightless than about 1 μm.